Mechanisms of cytokine effects on G protein-coupled receptor-mediated signaling in airway smooth muscle

Rodolfo M. Pascual1, Charlotte K. Billington2, Ian P. Hall2, Reynold A. Panettieri Jr.3, James E. Fish1, Stephen P. Peters1, and Raymond B. Penn4

1 Division of Critical Care, Pulmonary, Allergic, and Immunological Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University; 2 Department of Therapeutics, Institute of Cell Signaling, University Hospital of Nottingham, Nottingham, United Kingdom NG7 2UH; 3 Division of Pulmonary and Critical Care, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104; and 4 Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Numerous in vitro and in vivo studies have implicated the cytokines interleukin-1beta (IL-1beta ) and tumor necrosis factor-alpha (TNF-alpha ) as mediators of airway inflammation and therefore potentially important substances in the pathogenesis of asthma. In this study, we examined the mechanisms by which IL-1beta and TNF-alpha affect inhibition of cell growth, G protein-coupled receptor (GPCR) desensitization, and the recently reported adenylyl cyclase sensitization in human airway smooth muscle (HASM) cultures. Our findings demonstrate that adenylyl cyclase sensitization is independent of cytokine-mediated cyclooxygenase type 2 (COX-2) and prostaglandin E2 (PGE2) induction, whereas COX-2 induction appears to be required for both growth inhibition and GPCR desensitization. However, GPCR desensitization was highly dependent on the presence of EGF during chronic treatment with cytokines, which could be explained by a synergistic effect of EGF on cytokine-mediated COX-2 and PGE2 induction. Interestingly, various agents (including inhibitors of p42/p44 and p38 mitogen-activated protein kinase signaling) were significantly more effective in inhibiting cytokine-mediated PGE2 induction, GPCR desensitization, and cell growth inhibition than in inhibiting COX-2 induction. These data demonstrate disparity in the requirement and sufficiency of COX-2 induction in promoting different functional effects of IL-1beta and TNF-alpha in HASM.

interleukin-1beta ; tumor necrosis factor-alpha ; adenylyl cyclase; sensitization


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN BRONCHOALVEOLAR LAVAGE FLUID harvested from asthmatic subjects, interleukin-1beta (IL-1beta ) and tumor necrosis factor-alpha (TNF-alpha ) levels are elevated (8, 23). Several in vitro studies have demonstrated the effects of these cytokines on airway cells (including resident stromal cells and infiltrating blood cells) that are consistent with a role for IL-1beta and TNF-alpha as mediators of airway inflammation. These effects include increased adhesion-molecule expression, cytokine and matrix secretion, and cell activation and chemotaxis (reviewed in Refs. 1, 12, and 15).

In airway smooth muscle (ASM) cultures, chronic IL-1beta treatment has been shown to inhibit mitogen-induced cell growth (3) and promote hyporesponsiveness of beta 2-adrenergic receptors (beta 2-ARs) as defined by diminished inhibition of cell contraction (19, 21, 24, 33) and decreased cAMP production (14, 19, 21, 24, 28, 33) in response to beta -agonists. It has been suggested that these effects are mediated through an induction of cyclooxygenase type 2 (COX-2) expression and the associated increase in prostaglandin E2 (PGE2) production (3, 21). COX catalyzes the conversion of arachidonic acid to PGH2, which is subsequently converted to prostanoids including PGE2, PGI2, and thromboxane A2. Of the two principal COX isoforms, COX-1 appears to be constitutively expressed in most cell types and is responsible for prostanoid synthesis under physiological conditions, whereas COX-2 is induced by various inflammatory stimuli (reviewed in Ref. 21). In numerous cell types including ASM, IL-1beta treatment has been shown to increase COX-2 protein expression as well as cause accumulation of PGE2 in cell-culture media in an indomethacin-sensitive manner. Because treatment of ASM cultures with PGE2 also results in inhibition of ASM growth and beta 2-AR hyporesponsiveness (3, 21), these collective findings strongly implicate PGE2 generated as a result of COX-2 induction as the mediator of these cytokine-induced effects.

However, we have recently reported that chronic treatment of human ASM (HASM) cultures with IL-1beta also results in sensitization of adenylyl cyclase (AC) (5). Because AC has been suggested to represent the rate-limiting component in cAMP production mediated by the G protein-coupled receptor (GPCR)-Gs protein-AC transmembrane signaling cascade (4), sensitization appears to be a homeostatic mechanism that mitigates the deleterious effect of cytokines on beta 2-AR responsiveness to preserve beta -agonist-stimulated cAMP production (5).

In this study, we compare the mechanisms by which IL-1beta and TNF-alpha promote the induction of COX-2 and PGE2, inhibition of cell growth, GPCR desensitization, and AC sensitization in HASM cultures. Our findings demonstrate that AC sensitization is independent of cytokine-mediated COX-2 and PGE2 induction, whereas COX-2 induction appears to be required for both growth inhibition and GPCR desensitization. However, GPCR desensitization was highly dependent on the presence of mitogenic growth factors such as epidermal growth factor (EGF) during chronic treatment with cytokines, which could be explained by a synergistic effect of EGF on cytokine-mediated COX-2 and PGE2 induction. Interestingly, various agents [including inhibitors of p42/p44 and p38 mitogen-activated protein kinase (MAPK) signaling] were significantly more effective in inhibiting cytokine-mediated PGE2 induction, GPCR desensitization, and cell growth inhibition than in inhibiting COX-2 induction. These findings demonstrate disparity in the requirement and sufficiency of COX-2 induction in promoting different functional effects of IL-1beta and TNF-alpha in HASM.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. The Biotrak 125I-PGE2 RIA kit, ECL reagents, and [methyl-3H]thymidine (1 µCi/ml) were purchased from Amersham (Arlington Heights, IL). Human anti-COX-2 antibody was purchased from Oxford Biomedical Research (Oxford, MI). 125I-adenosine 3',5'-cyclic phosphoric acid (2,200 Ci/mmol) and [125I]iodopindolol (PIN; 2,200 Ci/mmol) were purchased from NEN-Dupont (Boston, MA). U-0126 was a gift from Dupont Pharmaceuticals (Wilmington, DE). cAMP antibody was provided by Mario Ascoli (University of Iowa, Iowa City, IA). SB-203580 and SB-202474 were purchased from Calbiochem (San Diego, CA). All other reagents were purchased from Sigma (St. Louis, MO) or from previously identified sources (26, 30).

HASM cell culture. HASM cultures were established as described by Panettieri et al. (27) from human tracheae obtained from lung-transplant donors in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. Characterization of this cell line with regard to immunofluorescence of smooth muscle actin and agonist-induced changes in cytosolic calcium has been previously reported (27).

Cells of passages 2-6 were plated at a density of 104 cells/cm2 in either 24-well ([3H]thymidine and cAMP assays) or 6-well (COX-2 protein, PGE2, and MAPK assays) plates in fetal bovine serum (FBS)-supplemented medium as described previously (26, 30). Seven days later, cells were growth-arrested by washing them with phosphate-buffered saline (PBS) and refeeding them with Ham's F-12 medium supplemented with 5 µg/ml each of insulin and transferrin (IT medium) for 48 h.

Assay of cAMP accumulation. HASM cultures were growth-arrested for 48 h as described and treated with vehicle or indicated inhibitors for 30 min before being treated with either 20 U/ml IL-1beta , 10 ng/ml TNF-alpha , or both in the presence or absence of 10 ng/ml EGF for 18 h. Cultures were then washed thoroughly with cold PBS and subsequently stimulated with 500 µl of PBS containing 300 µM ascorbic acid, 1 mM Ro-20-1724 (a phosphodiesterase inhibitor), and either vehicle (basal), 1 µM isoproterenol (Iso), 10 nM or 1 µM PGE2, or 100 µM forskolin (Fsk) for 10 min at 37°C. cAMP was isolated and quantified by radioimmunoassay using an 125I-cAMP tracer and cAMP antibody (31) as described previously (30).

Radioligand binding. To assess possible changes in beta 2-AR density and distribution in HASM cells mediated by chronic cytokine + EGF treatment, cells were grown to confluence in 15-cm dishes, growth-arrested for 48 h, then treated for 18 h with cytokine + EGF as described. Cells were washed in cold PBS and harvested as described previously (30). Approximately 100 µg of resuspended cells were incubated with 200 pM [125I]PIN in the presence or absence of 1 µM alprenolol (for determination of whole cell beta 2-AR density) or 100 nM CGP-12177 (cell-surface beta 2-ARs) at 14°C for 3.5 h, and reactions were terminated via filtration through Whatman GF-C filters using a Brandel cell harvester as described previously (30).

Assay of [3H]thymidine incorporation and cell proliferation. Confluent growth-arrested cells were pretreated with inhibitors or the matched vehicle and were stimulated with various agents as indicated. After 16 h of stimulation, cells in 24-well plates were labeled with 3.0 µCi [methyl-3H]thymidine (1 µCi/ml) and incubated at 37°C for 24 h. Cells were then washed with PBS, harvested with 0.05% trypsin in 0.53 mM EDTA, and lysed with 20% trichloroacetic acid. The precipitate was aspirated onto filter paper and counted in scintillation vials. Data points from individual [3H]thymidine experiments represent the mean values derived from six wells.

Analysis of COX-2 expression and PGE2 production. HASM cultures were grown in six-well plates and treated exactly as described for cAMP assays. After 18 h of treatment with cytokines, the plates were placed on ice and 250 µl of media were harvested from each well for subsequent analysis of PGE2 concentration. The rest of the media were then aspirated from the well, the well was washed once with cold PBS, and 200 µl of 1% sodium dodecyl sulfate (SDS) sample buffer (26) were applied directly to the well. Cell lysates were harvested by scraping and subsequently analyzed by immunoblotting for COX-2 expression using a 1:2,000 dilution of primary antibody. PGE2 from the harvested culture media was purified using C-18 columns as described previously (17) and subsequently quantified using the Biotrak RIA kit as per the manufacturer's instructions.

Analysis of p42/p44, p54 c-Jun amino-terminal kinase, and p38 MAPK phosphorylation. HASM cells were plated in six-well plates as described (see HASM cell culture) and stimulated with various agents for 0-18 h. At the indicated time points, cells were washed once with cold PBS and then lysed by addition of SDS sample buffer. Lysates were analyzed by immunoblotting using antibodies that specifically recognize the phosphorylated forms of p42/p44, p54 c-Jun amino-terminal kinase (JNK), and p38 MAPK, as described previously (26). To confirm uniformity of gel loading, blots were stained with 0.2% Ponceau S, or parallel blots were run and probed with antibodies that recognize both phosphorylated and nonphosphorylated forms of the respective MAPKs.

Data presentation and statistical analysis. Data points from individual assays represent the mean values from duplicate or triplicate measurements. Statistical analysis of data was performed using GraphPad Prism Software (San Diego, CA) software. Except where noted, data are presented as means ± SE. EGF- and cytokine-mediated effects on basal and agonist-stimulated cellular cAMP accumulation are reported in Fig. 2. Agonist-stimulated cellular cAMP production was calculated as the cAMP measured in agonist-treated cells minus the matched basal value. To minimize interexperimental variability in subsequent experiments that examined the effect of various inhibitory agents, group values were normalized to the appropriate matched control value (either vehicle-treated or vehicle + EGF-treated groups). For PGE2-stimulated group data, the data reflecting values for 10 nM PGE2 stimulation are presented. However, experimental effects on cells stimulated with 1 µM PGE2 were qualitatively similar (data not shown). Statistically significant differences among groups were assessed by either ANOVA with Fisher's protected least-significant difference post hoc analysis (StatView 4.5, Abacus Concepts, Berkeley, CA) or by t-test for paired samples with P < 0.05 sufficient to reject the null hypothesis.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of cytokine on cell growth, GPCR desensitization, and AC sensitization. Initial studies characterized the effects of chronic treatment with IL-1beta , TNF-alpha , or both on cell growth and GPCR-AC signaling in HASM cultures. Chronic IL-1beta , TNF-alpha , or IL-1beta  + TNF-alpha treatment had little or no effect on HASM [3H]thymidine incorporation (data not shown and Ref. 26). Consistent with previous observations noting the inhibitory effects of cytokines on serum-stimulated DNA synthesis in HASM cultures (3), EGF-stimulated DNA synthesis was significantly inhibited by IL-1beta or combined IL-1beta  + TNF-alpha (but not TNF-alpha alone) treatment, and these effects were reversed by prior treatment with 1 µM indomethacin, which suggests a dependence on COX activity (Fig. 1). On the basis of the results of previous studies (14, 28, 33) and our previous analyses of mechanisms of GPCR desensitization in HASM (25, 30) which would suggest that the cytokine-promoted induction of COX-2 and the associated PGE2 production should cause a desensitization of both the beta 2-AR and PGE2 receptors in ASM, we examined the responsiveness of these receptors (defined by agonist-stimulated cAMP generation) after chronic cytokine treatment. In contrast with previous studies (14, 28, 33), chronic treatment with IL-1beta did not decrease Iso- or PGE2-stimulated cAMP production relative to that stimulated in the control (vehicle-treated) group (Fig. 2, B and C). Similarly, chronic treatment with TNF-alpha failed to inhibit agonist-stimulated cAMP generation. Only combined treatment with IL-1beta and TNF-alpha resulted in a loss of Iso- and PGE2-stimulated cAMP generation. However, concomitant treatment with EGF rendered IL-1beta effective in inhibiting Iso- and PGE2-stimulated cAMP generation and also increased the inhibitory effect of IL-1beta  + TNF-alpha treatment. Cells cotreated with EGF and either IL-1beta or IL-1beta  + TNF-alpha exhibited significantly lower agonist-stimulated cAMP values than those of the matched vehicle-treated (with EGF) group (Fig. 2, B and C). In addition, cAMP values for the EGF + IL-1beta and EGF + IL-1beta  + TNF-alpha groups were also significantly lower than those of the vehicle-treated (without EGF) group for each agonist, with the exception being that Iso-stimulated cAMP values in the EGF + IL-1beta group were reduced (but not significantly so) relative to the vehicle-treated (with EGF) group (22.1 ± 2.1 vs. 18.5 ± 1.9 pmol/well, respectively). In contrast, EGF treatment failed to confer any desensitizing effect of TNF-alpha treatment. Despite the clear effects of the treatment conditions on DNA synthesis (measured after 40 h of treatment; see Fig. 1), protein content per well (measured after 18 h of treatment) did not vary significantly among the treatment groups (data not shown), which suggests that alterations in cell number or whole-cell protein content did not contribute to the observed experimental effects on cAMP production.


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Fig. 1.   Effect of cytokines on epidermal growth factor (EGF)-stimulated [3H]thymidine incorporation. Confluent growth-arrested human airway smooth muscle (HASM) cultures grown in 24-well plates were treated with vehicle (Veh) or indomethacin (Indo; 1 µM) for 30 min and then treated with 20 U/ml interleukin-1beta (IL-1beta ), 10 ng/ml tumor necrosis factor-alpha (TNF-alpha ), or both (I + T) in the presence and absence of 10 ng/ml EGF for 16 h. Cultures were labeled with 3.0 µCi of [methyl-3H]thymidine (1 µCi/ml) for 24 h and harvested for determination of [3H]thymidine incorporation as described in METHODS. Individual data points represent the mean values derived from 6 wells. Mean values were normalized to matched EGF + Veh control value, which was 65 ± 12-fold of basal incorporation and similar to previously reported values (16). Values are means ± SE; n = 5 experiments. *P < 0.05 for EGF Veh group vs. EGF + IL-1beta or EGF + IT group. **P < 0.05 for Indo vs. matched Veh EGF + IL-1beta or EGF + IT group.



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Fig. 2.   Regulation of G protein-coupled receptor (GPCR) adenylyl cyclase (AC) responsiveness by cytokines and EGF. Confluent growth-arrested HASM cultures grown in 24-well plates were treated with vehicle (CON), 20 U/ml IL-1beta , 10 ng/ml TNF-alpha , or IT in the presence and absence of 10 ng/ml EGF for 18 h. Cells were washed in cold phosphate-buffered saline (PBS) and challenged with basal Veh (A), 1 µM isoproterenol (Iso; B), 10 nM PGE2 (C), or 100 µM forskolin (Fsk; D) for 10 min at 37°C. cAMP was isolated and quantified by radioimmunoassay as described in METHODS. Data are means ± SE from 10-15 observations. *P < 0.05 for experimental vs. Veh no-EGF group values. **P < 0.05 for experimental vs. Veh + EGF group values.

Because AC is the downstream effector in GPCR-mediated cAMP production and its specific regulation can affect signaling (4), we also examined the effect of chronic cytokine treatment on cAMP production elicited by stimulation with Fsk, a diterpine characterized as a direct activator of AC (32). Interestingly, each of the treatments (IL-1beta , TNF-alpha , or both) caused a sensitization of AC defined by an increase in Fsk-stimulated cAMP production (Fig. 2D). This sensitization also occurred in cells cotreated with EGF. However, treatment with EGF alone also caused AC sensitization, and the combined effects of EGF and cytokines were less than additive, which suggests that EGF and cytokines promote AC sensitization via partially overlapping redundant mechanisms. Cytokine-mediated effects in basal cAMP levels (Fig. 2A) tended to parallel those observed on Fsk-stimulated cAMP generation (although absolute basal cAMP levels were typically <2% of Fsk-stimulated levels), which suggests that basal cAMP accumulation in cells reflects in part intrinsic AC activity.

To explore the potential role of receptor downregulation in the observed desensitization of the beta 2-AR, changes in beta 2-AR density were assessed in cells treated for 18 h with IL-1beta , TNF-alpha , or both, as well as EGF and EGF + IL-1beta . As shown in Fig. 3, only IL-1beta  + TNF-alpha , and EGF + IL-1beta caused significant decreases in beta 2-AR density (24 ± 6.5% and 30 ± 5% reductions, respectively, compared with vehicle-treated group; P < 0.05), which demonstrates that beta 2-AR downregulation is associated with those conditions that elicited beta 2-AR desensitization (Fig. 2). beta 2-AR distribution in HASM cells (~80% of total receptor number localized to cell surface) was not affected by any of the treatments (data not shown).


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Fig. 3.   Effect of cytokine treatment on beta 2-adrenergic receptor (beta 2-AR) density in HASM cells. Cells grown in 15-cm dishes were growth arrested and treated with CON, 20 U/ml IL-1beta , 10 ng/ml TNF-alpha , IL-1beta  + TNF-alpha (I + T), 10 ng/ml EGF, or EGF + IL-1beta for 18 h. Cells were washed in PBS and harvested, and beta 2-AR density was measured by incubating ~100 µg of cells in PBS containing 200 pM [125I]iodopindolol in the presence and absence of 1 µM alprenolol at 14°C for 3.5 h. Reactions were terminated by filtration through Whatman GF-C filters as described previously (15). CON beta 2-AR density = 21.4 ± 3.2 fmol/mg protein. Data are means ± SE of 4 experiments. *P < 0.05 for I + T, EGF + IL-1beta vs. matched CON group values.

Effect of COX inhibition on GPCR desensitization and AC sensitization. Prior treatment with indomethacin reversed the GPCR desensitization exhibited under both EGF-free and EGF-containing conditions, which again suggests a dependence on COX activity (Fig. 4, A-D). However, under all treatment conditions, cytokine-promoted AC sensitization was minimally affected by prior indomethacin (Fig. 4, E and F) or dexamethasone (data not shown) treatment, which suggests a COX-independent mechanism. Similarly, the effects of cytokine on basal cAMP levels were not altered by indomethacin treatment (data not shown).


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Fig. 4.   Effect of Indo on cytokine-mediated alterations in GPCR and AC responsiveness. Confluent growth-arrested HASM cultures grown in 24-well plates were treated with Veh or 1 µM Indo for 30 min then treated with 20 U/ml IL-1beta , 10 ng/ml TNF-alpha , or I + T in the presence and absence of 10 ng/ml EGF for 18 h. Cells were washed in cold phosphate-buffered saline (PBS) and challenged with 1 µM Iso (A and B), 10 nM PGE2 (C and D), or 100 µM Fsk (E and F) as described in Fig. 2. cAMP was isolated and quantified by radioimmunoassay as described in METHODS. Data are means ± SE from 5-7 paired observations. *P < 0.05 for cytokine-treated vs. matched control (Veh CON or Veh EGF) group values. **P < 0.05 for Indo vs. matched group (Veh IL-1beta , Veh I + T, EGF + IL-1beta , or EGF + I + T) values.

Effects of EGF on cytokine-mediated COX-2 and PGE2 induction. Given the potentiating effect of EGF on cytokine-mediated GPCR desensitization, we next examined the effects of EGF on COX-2 and PGE2 induction by cytokines. In the absence of EGF, COX-2 protein levels were undetectable in both vehicle- and TNF-alpha -treated cells. IL-1beta -induced COX-2 expression and combined IL-1beta  + TNF-alpha treatment typically increased COX-2 expression to a level more than twofold that elicited by IL-1beta treatment alone (Fig. 5A). Treatment with EGF alone had a small effect on COX-2 expression that was observable only upon overexposure of autoradiographs (data not shown). However, EGF significantly increased both IL-1beta - and IL-1beta  + TNF-alpha -induced COX-2 expression and permitted a small but observable induction of COX-2 expression by TNF-alpha . A similar potentiation of COX-2 synthesis was observed in HASM cells treated with a combination of cytokine(s) and either platelet-derived growth factor or 10% (final) FBS (data not shown).


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Fig. 5.   Effect of EGF on cytokine-mediated induction of cyclooxygenase type 2 (COX-2) and PGE2. Confluent growth-arrested HASM cultures grown in 6-well plates were treated with 20 U/ml IL-1beta , 10 ng/ml TNF-alpha , or I + T in the presence and absence of 10 ng/ml EGF for 18 h. Cell lysates were harvested for immunoblot analysis of COX-2 expression (A), and cell culture supernatants were analyzed for PGE2 content by radioimmunoassay as described in METHODS. Data are means ± SD from 2 (TNF-alpha without EGF) and 4 (all other conditions) paired observations (B). Bas, basal vehicle.

Consistent with the observed effects on COX-2 expression, PGE2 levels from cell culture media were significantly elevated after IL-1beta treatment and to a greater extent after treatment with IL-1beta  + TNF-alpha (Fig. 5B). Cotreatment with EGF increased the levels of both IL-1beta - and IL-1beta  + TNF-alpha -induced PGE2 production. Under all pretreatment conditions, prior treatment with indomethacin or dexamethasone eliminated the increase in PGE2 accumulation (data not shown).

Cytokine- and EGF-stimulated activation of p42/p44, p38, and p54 JNK MAPK. Because previous studies in various cell types [including HASM (19, 20)] have implicated roles for both p42/p44 and p38 in both the induction of COX-2 expression and related functional effects elicited by cytokines, we examined the time-dependent activation of the MAPK pathways by IL-1beta , TNF-alpha , IL-1beta  + TNF-alpha , EGF, and combined EGF + IL-1beta treatments. Acute activation (30 min) of p42/p44 tended to be comparable among cells stimulated with IL-1beta , TNF-alpha , IL-1beta  + TNF-alpha , EGF, or EGF + IL-1beta (Fig. 6A). At 3 h and 6 h, activation by IL-1beta or TNF-alpha was still detectable, and activation by IL-1beta , TNF-alpha , EGF, and EGF + IL-1beta was significantly greater. By 12 h and 18 h, a weak activation by IL-1beta and TNF-alpha was still detectable; activation by EGF and to a lesser extent EGF + IL-1beta was still prominent. For p38, 30 min of treatment with IL-1beta or TNF-alpha promoted p38 phosphorylation, but phosphorylation induced by EGF was minimal (Fig. 6B) as described previously (26). Combined IL-1beta  + TNF-alpha treatment resulted in a slightly greater phosphorylation of p38 than either IL-1beta or TNF-alpha alone, whereas the effect of EGF + IL-1beta was comparable to that of IL-1beta alone. At the 3 h and 6 h time points, p38 phosphorylation by either IL-1beta or TNF-alpha was minimal, but both IL-1beta  + TNF-alpha as well as EGF + IL-1beta continued to promote a strong phosphorylation of p38. Analysis of later time points failed to reveal any significant activation of p38 by any of the stimuli beyond basal levels. For p54 JNK, stimulation with IL-1beta , TNF-alpha , IL-1beta  + TNF-alpha , and EGF + IL-1beta all significantly increased p54 JNK phosphorylation at the 30-min time point, with IL-1beta  + TNF-alpha treatment promoting a slightly greater signal than either IL-1beta or TNF-alpha alone (Fig. 6C). No clear activation of p54 JNK was observed at later (>3 h) time points.


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Fig. 6.   Time-dependent activation of p42/p44, p38, and p54 c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) by cytokines and EGF in HASM. Cells grown to confluence in 6-well plates were growth arrested and stimulated with the indicated agents for 0-18 h. Cell lysates were harvested, and phosphorylation of p42/p44 (A), p38 (B), and p54 JNK (C) was assessed by immunoblotting with phospho-specific antibodies against the respective isoforms as described previously (16). Autoradiographs are representative of 3 independent experiments for each MAPK isoform.

Effects of p42/p44 and p38 MAPK inhibition on cytokine-mediated effects. To assess the requirement for p42/p44 and p38 MAPK activity, we examined the effects of inhibiting p42/p44 [using the mitogen-activated protein kinase/extracellular signal-regulated kinase MEK1/2 inhibitor U-0126 (13)] and p38 [using SB-203580 (9)] on cytokine-mediated effects in HASM. Prior treatment with 10 µM U-0126 or 1 µM SB-203580 significantly reversed the cytokine-mediated inhibition of Iso- and PGE2-mediated cAMP production (Fig. 7A-D) as did pretreatment with indomethacin (see Fig. 4) or dexamethasone (not shown). In reversing the observed GPCR desensitization, 1 µM U-0126 and 100 nM SB-203580 were ~50% as effective as 10 µM U-0126 and 1 µM SB-203580, respectively (data not shown). Interestingly, pretreatment with 1 µM SB-202474 [an analog of SB-203580 that lacks the ability to inhibit p38 (22)] was also able to partially reverse cytokine-mediated beta 2-AR- and PGE2-receptor desensitization. Conversely, U-0126, SB-203580, and SB-202474 failed to inhibit cytokine-promoted AC sensitization (Fig. 7, E and F). In fact, pretreatment with SB-203580 or SB-202474 alone caused a slight increase in Fsk-stimulated cAMP production. Cytokine-mediated effects on basal cAMP were largely unaffected by the inhibitors, although U-0126 and SB-203580 each caused partial inhibition of the increase in basal cAMP levels caused by IL-1beta treatment (data not shown).


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Fig. 7.   Effects of inhibitors of p42/p44 and p38 on cytokine-mediated alterations in GPCR and AC responsiveness. Confluent growth-arrested cultures grown in 24-well plates were pretreated with Veh (0.01% DMSO), 10 µM U-0126, 1 µM SB-203580 (SB203), or 1 µM SB-202474 (SB202) for 30 min then chronically treated with the indicated cytokines(s) ± EGF as described in Fig. 2. Cells were washed in cold PBS and challenged with 1 µM Iso (A and B), 10 nM PGE2 (C and D), or 100 µM Fsk (E and F) for 10 min at 37°C. cAMP was isolated and quantified by radioimmunoassay as described in METHODS. Data are means ± SE from 4-7 paired observations. *P < 0.05 for cytokine-treated group value vs. matched control (Veh CON or Veh EGF). **P < 0.05 for inhibitor group (IL-1beta or I + T + U-0126, SB203, or SB202, either no-EGF or + EGF) vs. matched group (Veh IL-1beta , Veh I + T, EGF + IL-1beta , or EGF + I + T) values.

SB-203580 was essentially as effective as indomethacin in reversing cytokine-mediated inhibition of EGF-stimulated [3H]thymidine incorporation, whereas SB-202474 was partially effective (Fig. 8).


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Fig. 8.   Effects of SB-203580 and SB-202472 on cytokine-mediated inhibition of HASM growth. Confluent growth-arrested cultures were pretreated with Veh (0.01% DMSO), 1 µM SB-203580, 1 µM SB202474, or 1 µM Indo for 30 min and then treated with 10 ng/ml EGF with or without the indicated cytokine(s). [3H]thymidine incorporation was subsequently determined as described in METHODS. Data are means ± SE from 4-6 experiments. *P < 0.05 for Veh EGF group vs. Veh EGF + IL-1beta or Veh EGF + I + T group. **P < 0.05 for inhibitor group (EGF + IL-1beta or EGF + I + T ± SB203, SB202, or Indo) vs. matched group (Veh EGF + IL-1beta or Veh EGF I + T) values.

Further analysis of the effects of these compounds on the induction of COX-2 and PGE2 revealed interesting findings. Both U-0126 and SB-203580 inhibited COX-2 induction by IL-1beta by >80% (Fig. 9). Inhibition of IL-1beta  + EGF- and IL-1beta  + TNF-alpha -induced COX-2 expression (Figs. 8A and 9A, respectively) was less profound; inhibition ranged from 60-80% in five separate experiments. U-0126 and SB-203580 were least effective in inhibiting COX-2 induction by IL-1beta  + TNF-alpha  + EGF (typically a 50-60% reduction observed in five experiments; Fig. 9B). Pretreatment with SB-202474 had no effect on COX-2 induction elicited by any agent(s).


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Fig. 9.   Effects of U-0126, SB-203580, and SB-202472 on cytokine-mediated COX-2 PGE2 induction. Confluent growth-arrested cultures were pretreated with vehicle (0.01% DMSO), 10 µM U-0126, 1 µM SB-203580, or 1 µM SB-202474 for 30 min and then treated for 18 h with the indicated cytokine(s) with and without EGF as described in Fig. 4. Cell lysates were harvested for immunoblot analysis of COX-2 expression (A and B) and cell culture supernatants were analyzed for PGE2 content by radioimmunoassay (C) as described in METHODS. Autoradiographs from A and B are representative of 5 independent experiments; data from C represent means ± SE from 4 experiments.

Analysis of PGE2 levels (Fig. 9C) sampled from the same wells assayed for COX-2 expression produced seemingly incongruous results with those presented in Fig. 9, A and B. For every condition in which U-0126 and SB-203580 essentially eliminated COX-2 induction (e.g., with IL-1beta treatment), PGE2 levels were reduced to control values. However, under conditions in which COX-2 induction was only partially inhibited (IL-1beta  + EGF, IL-1beta  + TNF-alpha , or IL-1beta  + TNF-alpha  + EGF), U-0126 or SB-203580 similarly reduced PGE2 levels to those of the control. Moreover, SB-202474, which had no effect on cytokine-mediated induction of COX-2, significantly inhibited cytokine-induced PGE2 production by ~75%. These data suggest that the induction of COX-2 by cytokines in HASM is not sufficient to promote PGE2 production (and the associated effects on GPCR responsiveness and cell growth), and that cytokines upregulate the activity of other elements that are important and potentially limiting in the synthesis of PGE2.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that in HASM, cytokines regulate receptor-mediated cAMP production via two independent mechanistically distinct processes (Fig. 10). Through the induction of COX-2 and PGE2 synthesis that occurs via pathways sensitive to p42/p44, p38, and nuclear factor (NF)-kappa B inhibition, cytokines (IL-1beta or IL-1beta  + TNF-alpha ) indirectly stimulate cAMP production via PGE2-receptor activation and thereby modulate cell functions such as growth. PGE2-receptor activation ultimately leads to homologous desensitization of PGE2 receptors and heterologous desensitization of beta 2-ARs. The induction of COX-2-PGE2 synthesis and GPCR desensitization by cytokines is highly dependent on costimulation with growth factors/mitogens such as EGF, which serves to promote a more robust and sustained activation of p42/p44 and p38 MAPK than that stimulated by cytokines alone. Through an undetermined mechanism that is dissociated from COX-2-PGE2 induction and is not dependent on p42/p44, p38, or NF-kappa B activation, cytokines (IL-1beta , TNF-alpha , or IL-1beta  + TNF-alpha ) promote the sensitization of AC, which serves to augment cAMP production and minimize the impact of GPCR desensitization on receptor-mediated cAMP production. AC sensitization by cytokines is minimally affected by concomitant EGF treatment, suggesting that EGF and cytokines may invoke similar partially redundant mechanisms to sensitize AC.


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Fig. 10.   Model of cytokine- and EGF-mediated signaling events that regulate GPCR and AC responsiveness in HASM. Cytokines IL-1beta and TNF-alpha coordinate with EGF to activate 2 distinct signaling pathways that can lead to the desensitization of GPCRs and the sensitization of AC. Chronic treatment of HASM with IL-1beta stimulates p42/p44 and p38 MAPK and nuclear factor (NF)-kappa B to induce COX-2 protein synthesis, which in turn promotes the production of PGE2. Conversely, treatment with TNF-alpha is not capable of significantly inducing COX-2 or PGE2 synthesis. An additional factor potentially regulated by IL-1beta , which is inhibited by SB-202474, also appears to be required for PGE2 induction. PGE2 acts in an autocrine fashion to stimulate cAMP production via Gs-coupled PGE2 receptors, which results in protein kinase A (PKA) activation and the regulation of various cellular processes such as cell growth. PGE2 receptor activation also ultimately leads to the homologous desensitization of PGE2 receptors and to the heterologous desensitization of the beta 2-AR, presumably via a PKA-dependent mechanism. Concomitant treatment of HASM cells with EGF or TNF-alpha in addition to IL-1beta causes a greater activation of p42/p44 and p38 MAPK, potentiates the induction of COX-2 and PGE2, and enhances the degree of GPCR desensitization. Concomitant treatment with EGF and TNF-alpha results in small induction of COX-2 protein that only minimally affects GPCR responsiveness. Through activation of an independent pathway that also requires new protein synthesis, chronic treatment of HASM with IL-1beta , TNF-alpha , or EGF induces a sensitization of AC, which is defined by increased cAMP production in response to receptor-independent activation of AC by Fsk. A lesser degree of cooperativity between IL-1beta , TNF-alpha , and EGF is observed in the sensitization of AC compared with that observed with COX-2-PGE2 induction. AC sensitization serves to mitigate the loss in GPCR-mediated cAMP production that might normally occur with GPCR desensitization. DEX, dexamethasone.

The general observations that cytokines induce COX-2 and PGE2 synthesis, modulate ASM growth, and promote GPCR desensitization have been demonstrated by numerous studies. Previous studies using ASM cultures from various species have identified IL-1beta -mediated inhibition of serum-stimulated growth (3) as well as decreased beta 2-AR or PGE2-receptor-mediated cAMP production (14, 19, 21, 24, 28, 33) and inhibition of cell contraction and tension (19, 21, 24, 33). The role of COX-2 in mediating the effects of IL-1beta in inhibiting serum-stimulated ASM growth and promoting beta 2-AR hyporesponsiveness is supported by detailed analyses by the Knox laboratory of IL-1beta -mediated induction of COX-2 protein and activity, and associated eicosanoid synthesis in both bovine (10) and human (29) ASM cultures. Additional studies assessing IL-1beta effects on ASM growth (3) and GPCR signaling and function (21, 24, 28, 33) in ASM have also demonstrated IL-1beta -mediated COX-2 and PGE2 induction. In addition, IL-1beta -mediated growth inhibition, GPCR hyporesponsiveness, and COX-2 induction were all inhibited by glucocorticoid treatment (2, 24, 28, 34), and functional effects as well as PGE2 induction were similarly inhibited by indomethacin pretreatment (3, 21, 28). Moreover, pharmacological inhibition of p42/p44 and p38 MAPK pathways was shown to inhibit IL-1beta -mediated COX-2 and PGE2 synthesis and to reverse the associated loss of Iso-mediated relaxation of ASM stiffness (19, 20). Because exogenous PGE2 could mimic the effects of IL-1beta treatment, the collective evidence strongly implicates induced COX-2-mediated PGE2 synthesis as the principal mechanism by which IL-1beta alters ASM growth and GPCR function.

The present study extends these observations and provides new insight into mechanisms underlying cytokine-promoted COX-2 and PGE2 synthesis and the associated effects on GPCR responsiveness and cell growth. An important new finding relates to the sensitivity of cytokine-mediated effects to additional signaling input from growth factors. This sensitivity was originally suggested by Belvisi et al. (3), who reported that PGE2 release after chronic IL-1beta treatment was observed only in serum-fed (but not serum-deprived) HASM cells. In a recent study, we also noted that chronic IL-1beta treatment resulted in a loss of Iso-stimulated cAMP production in serum-fed but not serum-deprived HASM (5). Our current findings demonstrate that IL-1beta is not sufficient to induce COX-2 and PGE2 such that GPCR-mediated cAMP production is significantly inhibited, and requires additional signaling input from either growth factors or TNF-alpha to promote such an effect.

The insufficiency of IL-1beta to attenuate GPCR-mediated cAMP production is further explained by the sensitization or superactivation of AC that occurs not only with treatment with IL-1beta , but also with TNF-alpha . Although a desensitization of beta 2-AR and PGE2 receptors caused by IL-1beta treatment (in the presence or absence of EGF) is more readily interpreted if one considers the accompanying sensitization of AC (and assumes that increased intrinsic AC activity proportionally increases Gs-coupled receptor-mediated cAMP generation), the functional consequence of this desensitization (reduced cAMP generation in response to Iso or PGE2) is effectively reversed as a result of AC sensitization. Our finding of IL-1beta - and TNF-alpha -promoted AC sensitization is at odds with findings from numerous other studies that have reported no effect of either chronic IL-1beta (14, 21, 24, 33) or TNF-alpha (11, 16) treatment on AC responsiveness. Differences in species or culture conditions may explain the disparity between our results and those of others. Hakonarson et al. (14) examined rabbit ASM strips in which different (muscarinic-receptor dependent and pertussis-toxin sensitive) mechanisms appeared to be involved in mediating the effects of cytokines on GPCR signaling. Emala et al. (11) observed no effect of 72 h of TNF-alpha treatment on AC activity in canine ASM, although a subsequent study from the same group noted a trend toward increased AC activity (~46% mean increase in five experiments) caused by 72 h of TNF-alpha treatment of HASM (16). Studies from the Shore (33) laboratory focus on examining regulation of cell stiffness in HASM cultures, and parallel analyses of GPCR-AC-stimulated cAMP production are performed under carefully matched conditions in which cells are harvested after chronic cytokine treatment and replated shortly before performance of cAMP assays. Given the sensitivity of cytokine-dependent effects to growth factor (and other mitogenic) stimuli that is demonstrated in the present study, such conditions may obscure detection of AC sensitization.

Interestingly, this sensitization of AC was only minimally inhibited by pertussis toxin [which suggests a mechanism distinct from that responsible for AC elicited by Gi-coupled receptors (4)] and was also minimally affected by indomethacin (the present study), which demonstrates that this phenomenon is not dependent on COX activity. As noted above, the observed sensitization of AC by EGF treatment alone and the less-than-additive effect of concomitant EGF and cytokine treatment suggest that EGF and cytokines may share, to some degree, a common mechanism that promotes AC sensitization. However, inhibition of the mitogen-activated kinases p42/p44 (by U-0126) or p38 (by SB-203580) did not inhibit AC sensitization caused by either EGF or cytokines, although it was highly effective in inhibiting COX-2 induction, PGE2 production, and GPCR desensitization. Importantly, these latter positive effects of both U-0126 and SB-203580 occurred at inhibitor concentrations (10 µM and 1 µM, respectively) that favor specificity toward the intended targets (p42/44 and p38). Particularly problematic may be the nonspecific effects of SB-203580, which can include direct inhibition of COX-1 and COX-2 (7), thromboxane A2 synthase (7), or phosphoinositide-dependent protein kinase 1 (18), or enhancement of NF-kappa B activity (6) when used at concentrations >2 µM in intact cell experiments.

Last, our findings suggest an apparent insufficiency of COX-2 induction to promote significant PGE2 production and the associated effects on ASM growth and GPCR desensitization. Analysis of the relative effects of U-0126, SB-203580, and SB-202474 on COX-2 versus PGE2 induction supports this assertion. We noted a significant level of COX-2 protein was still induced by treatment with IL-1beta  + TNF-alpha , IL-1beta  + EGF, or IL-1beta  + TNF-alpha  + EGF when cells were pretreated with U-0126 or SB-203580, yet the accompanying PGE2 production was essentially eliminated by these agents. One interpretation of these results is that a very high threshold level of COX-2 induction is required before PGE2 production and the functional effects elicited by cytokines can occur. However, this does not appear to be the case, because IL-1beta induces a similar level of COX-2 induction compared with that induced by IL-1beta  + EGF in the presence of U-0126 or SB-203580 but causes a much greater induction of PGE2. Moreover, SB-202474, the analog of SB-203580 that lacks the ability to inhibit p38, causes no inhibition of cytokine-induced COX-2 yet inhibits PGE2 production by ~75%. These findings challenge the dogma that COX-2 is limiting and is the critical factor in the production of PGE2 in ASM, and suggest that the activity of another element in the pathway that promotes increased PGE2 synthesis is upregulated and that this activity can be inhibited by agents that target p42/p44 (U-0126) or some other molecule (that is inhibited by U-0126, SB-203580, or SB-202474) to inhibit the functional consequences of cytokines on ASM. Within this context, the role for U-0126, SB-203580, and SB-202474 in directly inhibiting enzyme activity or the induction of enzymes, cofactors, or possible complex formation important to PGE2 synthesis remains to be established and represents an interesting challenge for future studies.


    ACKNOWLEDGEMENTS

We acknowledge Kristin Brodbeck and Andrew Eszterhas for valued technical assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-58506 (to R. B. Penn) and HL-64063 (to R. A. Panettieri) and National Institute of Allergy and Infectious Diseases Grant AI-24509 (to S. P. Peters).

R. M. Pascual is recipient of a Glaxo Wellcome Pulmonary Fellowship, a Merck Young Investigator Award, and a Parker B. Francis Fellowship. R. B. Penn is a recipient of the American Lung Association Career Investigator Award.

Address for reprint requests and other correspondence: R. B. Penn, Thomas Jefferson Univ., Kimmel Cancer Institute, Rm. 930 BLSB, 233 S. 10th St., Philadelphia, PA 19107 (E-mail: rpenn{at}lac.jci.tju.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 16 January 2001; accepted in final form 3 August 2001.


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DISCUSSION
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