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Am J Physiol Lung Cell Mol Physiol 292: L1414-L1421, 2007. First published February 2, 2007; doi:10.1152/ajplung.00123.2006
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Raf-1 kinase mediates adenylyl cyclase sensitization by TNF-{alpha} in human airway smooth muscle cells

Yoko Osawa,1 Peter D. Yim,1 Dingbang Xu,1 Reynold A. Panettieri,2 and Charles W. Emala1

1Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York; and 2Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Submitted 3 April 2006 ; accepted in final form 28 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tumor necrosis factor (TNF)-{alpha} is a potent inflammatory cytokine implicated in the exacerbation of asthma. Chronic exposure to TNF-{alpha} has been reported to induce G protein-coupled receptor desensitization, but adenylyl cyclase sensitization, in airway smooth muscle cells by an unknown mechanism. Cyclic AMP, which is synthesized by adenylyl cyclases in response to G protein-coupled receptor signals, is an important second messenger involved in the regulation of the airway muscle proliferation, migration, and tone. In other cell types, TNF-{alpha} receptors transactivate the EGF receptor, which activates raf-1 kinase. Further studies in transfected cells show that raf-1 kinase can phosphorylate and activate some isoforms of adenylyl cyclase. Cultured human airway smooth muscle cells were treated with TNF-{alpha} in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, or Gi proteins. TNF-{alpha} caused a significant dose- (1–10 ng/ml) and time-dependent (24 and 48 h) increase in forskolin-stimulated adenylyl cyclase activity, which was abrogated by pretreatment with GW5074 (a raf-1 kinase inhibitor), was partially inhibited by an EGF receptor inhibitor, but was unaffected by pertussis toxin. TNF-{alpha} also increased phosphorylation of Ser338 on raf-1 kinase, indicative of activation. IL-1beta and EGF sensitization of adenylyl cyclase activity was also sensitive to raf-1 kinase inhibition by GW5074. Taken together, these studies link two signaling pathways not previously characterized in human airway smooth muscle cells: TNF-{alpha} transactivation of the EGF receptor, with subsequent raf-1 kinase-mediated activation of adenylyl cyclase.

GW5074; mitogen-activated protein kinase; phosphorylation; immunoblot; cell culture

ASTHMA TYPICALLY INDUCES REVERSIBLE airway obstruction and is orchestrated in part by a complex interplay between many inflammatory cytokines (18, 24, 27). Traditionally, the influx of leukocytes with secretion of cytokines during asthma exacerbations has received the most attention, but multiple cells within the airway respond to, and themselves synthesize, cytokines, including airway smooth muscle (24). Tumor necrosis factor (TNF)-{alpha} is one cytokine implicated in the exacerbation of asthma (3, 6, 10), and recent clinical studies raise hope that targeted therapy against TNF-{alpha} may improve asthma symptoms (6). Cyclic AMP is an important second messenger involved in the regulation of the contractile state, proliferation (8, 25, 34), migration (19), and cytokine secretory abilities (1) of airway smooth muscle cells. Previous studies in human airway smooth muscle cells have shown that cytokines, such as TNF-{alpha} and IL-1beta, directly influence cellular cAMP levels by "sensitizing" the activities of adenylyl cyclase enzymes (9, 29), but the intracellular signaling pathways for the modification of adenylyl cyclase activity are unknown.

Cyclic AMP is synthesized by a nine-member family of membrane-associated adenylyl cyclase enzymes. These family members are differentially regulated both acutely and chronically. Acute regulation by phosphorylation by protein kinase A (PKA) and protein kinase C (PKC) or by G protein beta{gamma}-subunits has been most extensively characterized. Chronic regulation leading to increased enzyme activity has been most commonly demonstrated following chronic activation of Gi-coupled receptors, which, via raf-1 kinase-mediated phosphorylation, leads to increased enzyme activity and has been variably referred to as "sensitization," "supersensitization," "cAMP overshoot," "supersensitivity," "superactivation," or "heterologous sensitization" (36, 37).

Sensitization of adenylyl cyclase in airway smooth muscle cells has been reported following chronic exposure to agonists for receptors that couple to Gi (7, 26) cytokines (9, 29) or following rhinovirus exposure (9). In airway smooth muscle cells, all chronic Gi-coupled receptor-mediated sensitization of adenylyl cyclase has been blocked by pertussis toxin (PTX) (7, 26), consistent with a Gi -> raf-1 kinase-mediated sensitization pathway reported in other cell types following chronic opioid receptor activation (13, 35, 36). However, the role for raf-1 kinase in mediating adenylyl cyclase sensitization in airway smooth muscle cells has not been directly demonstrated. Moreover, the kinases potentially involved in cytokine-mediated (i.e., TNF-{alpha}, IL-1beta) sensitization of adenylyl cyclase in airway smooth muscle cells are unknown.

Understanding the mechanisms of cytokine-induced alterations in signal transduction pathways in human airway smooth muscle is important, as cytokines are an important component of the inflammatory response in asthmatic airways. In some cells, the activation of the TNF-{alpha} receptor is known to transactivate the EGF receptor (EGFR), which, in turn, can activate raf-1 kinase (2, 12). In unrelated experiments in transfected human embryonic kidney-293 cells, EGF activation of raf-1 kinase mediates phosphorylation and activation of adenylyl cyclase (5). We questioned whether the cellular signaling mechanism of TNF-{alpha}-mediated sensitization of adenylyl cyclase activity in native human airway smooth muscle cells involved the transactivation of the EGFR, resulting in raf-1 kinase-mediated adenylyl cyclase sensitization.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Materials. Cells were cultured in SmGM-2 smooth muscle medium (Cambrex, Walkersville, MD). Trypan blue stain 0.4% solution was obtained from Gibco (Grand Island, NY). [{alpha}-32P]ATP (800 Ci/mmol) and [3H]cAMP (32 Ci/mmol) were obtained from MP Biomedicals (Irvine, CA). Recombinant human TNF-{alpha}, PD98059, the EGFR/erbB-2 inhibitor [4-(4-benzyloxyanilino)-6,7-dimethoxyquinazoline, 4557W], and the monoclonal antibody (MAb) directed against the EGFR (MAb225) were obtained from Calbiochem (San Diego, CA). All other chemicals were obtained from Sigma (St. Louis, MO).

Cell culture. Primary cultures of human airway smooth muscle cells were obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings, as previously described (28). The cells were grown to confluence on 24-well or 6-well plates in culture medium (SmGM-2, supplemented with 5% FBS, 5 µg/ml insulin, 1 ng/ml human fibroblast growth factor, 500 pg/ml human EGF, 30 µg/ml gentamicin, and 15 ng/ml amphotericin B, Cambrex) at 37°C in 5% CO2-95% air. In all studies, EGF was omitted from the media at least 10 days before the beginning of treatments. Media containing serum and growth factors were changed twice weekly, but not changed for the 72 h immediately preceding 48-h treatments with TNF-{alpha} (72 h of media conditioning before 48-h TNF treatments).

Treatment protocols. Preliminary studies revealed an enhanced effect of TNF-{alpha} on forskolin-stimulated adenylyl cyclase sensitization in 72-h conditioned media. Therefore, in all studies, cell culture media (supplemented SmGM-2 devoid of EGF) were not changed for 72 h before the beginning of treatment with 10 ng/ml TNF-{alpha}. Preliminary experiments were performed to determine the optimum time and dose effect of TNF-{alpha} treatments. Forskolin-stimulated adenylyl cyclase activity was measured after 7, 24, or 48 h of TNF-{alpha} with redosing of TNF-{alpha} at 24 h for the 48-h time point. Subsequently, the dose effect (0.01–10 ng/ml) TNF-{alpha} was assayed for 48 h. Based on these preliminary studies, all subsequent TNF-{alpha} incubations were at 10 ng/ml for 48 h with redosing of TNF-{alpha} at 24 h.

In some studies, an inhibitor of prostaglandin synthesis, indomethacin (10 µM), was added 30 min before the beginning of 48-h TNF-{alpha} treatments. These inhibitors were also redosed at 24 h. In some studies, cells were pretreated with PTX 4 h before the beginning of 48-h TNF-{alpha} treatments.

Preincubation with the kinase inhibitors specific for PKC (100 nM GF109203X), mitogen-activated protein (MAP) kinase kinase (MEK) (50 µM PD98059), p38 MAP kinase (MAPK) (10 µM SB203580), tyrosine kinases (20 µM genistein), phosphatidylinositol 3-kinase (500 nM wortmannin), or raf-1 kinase (10 µM GW5074) were initially attempted for 30 min before the beginning of the 48-h TNF-{alpha} treatment. However, cell toxicity was evident with all kinase inhibitors during this prolonged incubation time, except for GW5074 (data not shown). Therefore, for relevant time point comparisons, all kinase inhibitors (GF109203X, PD98059, SB203580, genistein, wortmannin, GW5074) were added to the cells during the final 6 h or during the final 1 h of the 48-h TNF-{alpha} incubation. Additionally, in separate experiments, GW5074 was preincubated for 30 min before the addition of TNF-{alpha}. In independent studies, IL-1beta (0.5 ng/ml) or EGF (1 ng/ml) was incubated in the absence or presence of 10 µM GW5074 for 48 h. GW5074 was preincubated for 30 min before the addition of either IL-1beta or EGF, and both the ligand and inhibitor were redosed at 24 h. In some studies, an inhibitor of EGFR tyrosine kinase (EGFR/erbB-2; 10 µM) or MAb (MAb225; 2 µg/ml) that functionally blocks the EGFR was added 30 min or 1 h before the beginning of 48-h TNF-{alpha} or EGF treatments, respectively. These inhibitors were also redosed at 24 h along with TNF-{alpha} or EGF.

Adenylyl cyclase assays. Adenylyl cyclase activity was measured as previously described (30). Briefly, following 48-h treatments, with or without TNF-{alpha}, IL-1beta, or EGF in the presence or absence of selected inhibitors, cells in 24-well plates were washed once with warm PBS (37°C). One hundred microliters of warm PBS were added to each well. Subsequently, 50 µl of 3x adenylyl cyclase buffer (21) were added directly to the wells (to achieve final concentrations of 50 mM HEPES, pH 8.0, 50 mM NaCl, 0.4 mM EGTA, 1 mM cAMP, 7 mM MgCl2, 0.1 mM ATP [20 µCi/ml 32P-{alpha}-ATP], 0.1 mg/ml BSA, 50 U/ml creatine phosphoskinase, and 7 mM phosphocreatine) in the absence or presence of 10 µM forskolin, and plates were incubated at 37°C for 15 min. The reactions were terminated by the addition of 100-µl stop buffer [50 mM HEPES, pH 7.5, 2 mM ATP, 0.5 mM cAMP (0.5 µCi/ml 3H-cAMP), 2% SDS], and synthesized 32P-cAMP was separated and quantitated by sequential column chromatography over dowex and alumina, as described previously (30).

Immunoblotting for total or phosphorylated raf-1 kinase. Following 0-, 0.5-, 4-, 24-, or 48-h treatment with TNF-{alpha} (10 ng/ml), whole cell lysates were electrophoresed (10% SDS-PAGE) and immunoblotted using antibodies directed against phospho-raf-1 serine 338 (Ser338; 1:1,000; Upstate Cell Signaling Solutions, Charlottesville, VA) and total raf-1 (1:1,000; Santa Cruz, Santa Cruz, CA). In separate experiments, cells were pretreated with an inhibitor of the EGFR {EGFR/erbB-2 inhibitor [4-(4-benzyloxyanilino)-6,7-dimethoxyquinazoline, 4557W]} (10 µM) for 30 min before 24 h of TNF-{alpha} treatment. Epitopes were visualized with horseradish peroxidase-conjugated secondary antibodies (1:5,000; Amersham Biosciences, Arlington Heights, IL) using ECL Plus (Amersham Biosciences) and developed on film (Kodak BioMax light film, Kodak, Rochester, NY). Film was developed such that band intensities were within the linear range of film responses, and band intensities were quantified using Quantity One software (Bio-Rad, Hercules, CA). Levels of phosphorylated raf-1 kinase were expressed as ratios of total raf-1 kinase. Data presented are means ± SE values.

Immunoblotting for total adenylyl cyclase. Following 48-h treatment with TNF-{alpha} (10 ng/ml), whole cell lysates were electrophoresed (10% SDS-PAGE) and immunoblotted using an antibody directed against a 14-amino acid peptide of the COOH-terminal region of adenylyl cyclase, common to all cloned isoforms (1:2,000; Santa Cruz) or beta-actin (1:2,000, Sigma). Epitopes were visualized with horseradish peroxidase-conjugated secondary antibodies, as described above. Levels of immunoreactivity were normalized to beta-actin to control for total loaded protein. Data presented are means ± SE values.

Cell viability. Cell viability following TNF-{alpha} (10 ng/ml) exposure for 48 h in the presence or absence of GW5074 (10 µM) was measured in triplicate by Trypan blue dye exclusion. After incubation for 5 min with 0.2% Trypan blue in PBS, 300 cells in each group were counted, and the cells that internalized the dye were considered nonviable.

Statistics. Statistical analysis was performed using repeated-measures of ANOVA, followed by Bonferroni posttest comparison using Prism 4.0 software (GraphPad, San Diego, CA). Data are presented as means ± SEM; P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Adenylyl cyclase activity following TNF-{alpha} treatment of human airway smooth muscle cells. TNF-{alpha} demonstrated both a concentration-dependent and time-dependent sensitization of forskolin-stimulated adenylyl cyclase activity. Significant sensitization of forskolin-stimulated adenylyl cyclase activity was achieved at 24 h of TNF-{alpha} (10 ng/ml) treatment (Fig. 1A). Sensitization of forskolin-stimulated adenylyl cyclase activity occurred with concentrations of TNF-{alpha} ranging from 1 to 10 ng/ml, with a peak effect at 10 ng/ml (441 ± 86% of control) (Fig. 1B). Therefore, subsequent experiments were performed using 10 ng/ml TNF-{alpha}.


Figure 1
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  		Fig. 1. A: time course of 10 µM forskolin-stimulated adenylyl cyclase (AC) activity in cultured human airway smooth muscle cells. Cells were treated with tumor necrosis factor (TNF)-{alpha} (10 ng/ml) for the indicated times. B: 10 µM forskolin-stimulated AC activity in cultured human airway smooth muscle cells treated with TNF-{alpha} at indicated concentrations for 24 h. Data represent means ± SE; n = 4. Within each experiment, values were determined in triplicate. ***P < 0.001 compared with control.

 
Effect of indomethacin on TNF-{alpha}-induced adenylyl cyclase sensitization. Cells were pretreated with indomethacin (10 µM) for 30 min before 48 h of TNF-{alpha} treatment to determine whether the sensitization of adenylyl cyclase activity by TNF-{alpha} was dependent on TNF-{alpha}-induced prostaglandin release. Pretreatment with 10 µM indomethacin did not block the increase of forskolin-stimulated adenylyl cyclase activity induced by TNF-{alpha} (Fig. 2). The vehicle or indomethacin itself did not affect forskolin-stimulated adenylyl cyclase activity. These results suggest that sensitization of adenylyl cyclase activity by TNF-{alpha} is not mediated by prostaglandin release in these cells.


Figure 2
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  		Fig. 2. 10 µM forskolin-stimulated AC activity in cultured human airway smooth muscle cells following 48-h TNF-{alpha} (10 ng/ml) in the presence or absence of indomethacin (Ind) (10 µM 30 min before TNF-{alpha}). cont, Control. Data represent means ± SE; n = 3. Within each experiment, values were determined in triplicate. ***P < 0.001 compared with control.

 
Effect of Gi{alpha} on TNF-{alpha}-induced adenylyl cyclase sensitization. Adenylyl cyclase sensitization has classically been described following the chronic activation of Gi{alpha}-coupled receptors (37). To determine whether TNF-{alpha}-induced sensitization of adenylyl cyclase involved Gi proteins, human airway smooth muscle cells were pretreated with PTX (inactivates Gi/o proteins by ADP-ribosylation). PTX pretreatment had no effect on the TNF-{alpha}-induced sensitization of adenylyl cyclase in these human cultured airway smooth muscle cells (Fig. 3).


Figure 3
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  		Fig. 3. 10 µM forskolin-stimulated AC activity in cultured human airway smooth muscle cells following 48-h TNF-{alpha} (10 ng/ml) treatment in the presence or absence of pertussis toxin (PTX) (100 ng/ml for 4 h before 48-h TNF-{alpha} treatment). Data represent means ± SE; n = 5. Within each experiment, values were determined in triplicate. ***P < 0.001 compared with control.

 
Effect of kinase inhibitors on TNF-{alpha}-induced adenylyl cyclase sensitization. Multiple protein kinases, notably PKA, PKC, and raf-1 kinase, are known to affect adenylyl cyclase activity via subtype-specific phosphorylation (11, 17, 33). Raf-1 kinase has been reported to mediate the sensitization of adenylyl cyclase in a PTX-sensitive manner following chronic Gi{alpha} activation (17). Additionally, cytokines such as TNF-{alpha} are known to activate a myriad of intracellular kinase pathways (4, 22). Therefore, we questioned whether pretreatment with specific kinase inhibitors could attenuate TNF-{alpha}-induced adenylyl cyclase sensitization. Inhibitors of PKC, MEK, p38 MAPK, tyrosine kinases, phosphatidylinositol 3-kinase, or raf-1 kinase (GF109203X, PD98059, SB203580, genistein, wortmannin, GW5074, respectively) were added during the final 1 h (Fig. 4A) or the final 6 h (Fig. 4B) of the 48-h TNF-{alpha} incubation.


Figure 4
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  		Fig. 4. A: 10 µM forskolin-stimulated AC activity in cultured human airway smooth muscle cells following 48-h TNF-{alpha} (10 ng/ml) treatment in the presence or absence of kinase inhibitors: GW5074 (GW; 10 µM), PD98059 (PD; 50 µM), wortmannin (Wort; 500 nM), GF109203X (GF; 100 nM), SB203580 (SB; 10 µM), and genistein (Geni; 20 µM). Inhibitors were added the last 1 h (A) or 6 h (B) of TNF-{alpha} treatment. Data represent means ± SE; n = 5. Within each experiment, values were determined in triplicate. *P < 0.05 compared with control. #P < 0.05 compared with TNF.

 
The addition of the raf-1 kinase inhibitor GW5074 (10 µM) during the final 1 h (Fig. 4A) or the final 6 h (Fig. 4B) of the 48-h TNF-{alpha} treatment period attenuated the TNF-{alpha}-induced sensitization of adenylyl cyclase activity. This is in contrast to the lack of effect of all other kinase inhibitors tested at these 1- and 6-h time points (Fig. 4). Addition of the kinase inhibitors alone for either 1 or 6 h had no effect on forskolin-stimulated adenylyl cyclase activity (data not shown). In separate studies, the raf-1 kinase inhibitor GW5074 (10 µM) was added 30 min before 48 h of TNF-{alpha}, which completely reversed the TNF-{alpha}-induced sensitization of adenylyl cyclase activity (Fig. 5A). GW5074 alone for 48.5 h had no effect on forskolin-stimulated adenylyl cyclase activity. GW5074 pretreatment also blocked adenylyl cyclase sensitization induced by 48-h exposure to IL-1beta (Fig. 5B) and EGF (Fig. 5C).


Figure 5
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  		Fig. 5. 10 µM forskolin-stimulated AC activity in cultured human airway smooth muscle cells following 48-h TNF-{alpha} (10 ng/ml, n = 4; A), IL-1beta (500 pg/ml, n = 3; B), or EGF (1 ng/ml, n = 7; C) treatment in the presence or absence of GW5074 (10 µM for 30 min before each 48-h treatment). Data represent means ± SE. Within each experiment, values were determined in triplicate. **P < 0.01, ***P < 0.001 compared with control. #P < 0.05, ###P < 0.001 compared with TNF, IL-1beta, or EGF.

 
Effect of Raf-1 kinase inhibitor on cell viabilities. Pretreatment with 10 µM GW5074 for 48.5 h did not affect cell viabilities of the cells treated with TNF-{alpha}, as detected by Trypan blue exclusion. Viabilities of human airway smooth muscle cells treated with vehicle, 48-h TNF-{alpha} (10 ng/ml), or 48-h TNF-{alpha} plus 48.5-h GW5074 (10 µM) were 81.2 ± 4.4, 78.4 ± 4.1, or 78.0 ± 2.6%, respectively (P > 0.05, n = 5). These results suggest that TNF-{alpha}-induced adenylyl cyclase sensitization is mediated by raf-1 kinase.

Effect of TNF-{alpha} on phosphorylation of raf-1 kinase. Phosphorylation of Ser338 on the raf-1 kinase protein is indicative of kinase activation, which, in turn, is known to sensitize certain isoforms of adenylyl cyclase (15, 16). Therefore, we directly measured phosphorylation of Ser338 on raf-1 kinase following TNF-{alpha} treatment for 24 and 48 h in human airway smooth muscle cells. Twenty-four or 48 h of TNF-{alpha} treatment significantly increased phosphorylation of Ser338 on raf-1 kinase (Fig. 6, A and B), with no change in total raf-1 kinase. In separate experiments, pretreatment of cells with an inhibitor of the EGFR before 24 h of TNF-{alpha} treatment significantly attenuated the phosphorylation of raf-1 kinase (Fig. 6C).


Figure 6
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  		Fig. 6. A: Western blot analysis of phospho-Raf-1 or total Raf-1 expression in cultured human airway smooth muscle cells. Cells were treated with TNF-{alpha} (10 ng/ml) for the indicated times. A representative of three experiments is shown. B: relative band intensities of phospho-/total Raf-1 from three separate immunoblots. The effect of 24-h and 48-h treatment with TNF-{alpha} (10 ng/ml) on Raf-1 phosphorylation is given as percentage of control (n = 5). C: relative band intensities of phospho-/total Raf-1 illustrating the effect of pretreatement with an EGF receptor (EGFR) inhibitor (10 µM, 30 min) before 24-h treatment with TNF-{alpha} (10 ng/ml; n = 3). Values are means ± SE. *P < 0.05, **P < 0.01 compared with control. #P < 0.05 compared with 24-h TNF-{alpha}.

 
Effect of EGFR activation on TNF-{alpha}-induced adenylyl cyclase sensitization. EGFR transactivation has been reported to mediate TNF-{alpha} effects in various cell types (2, 12). To investigate the potential role of EGFR signaling in the adenylyl cyclase sensitization induced by TNF-{alpha}, adenylyl cyclase activity was measured in the presence or absence of a specific inhibitor of EGFR/erbB-2 tyrosine kinase or using a MAb directed against the EGFR. Pretreatment with the EGFR inhibitor partially blocked TNF-{alpha} effect on adenylyl cyclase activity (Fig. 7A). The EGFR inhibitor also inhibited EGF-induced adenylyl cyclase sensitization, indicating that this agent successfully inhibited the EGF signaling pathway (Fig. 7B). These results suggest that TNF-{alpha}-mediated transactivation of the EGFR could be at least one mechanism of TNF-{alpha}-induced adenylyl cyclase sensitization. In contrast, pretreatment with 2 µg/ml of EGFR antibody (MAb225) did not block the increase of forskolin-stimulated adenylyl cyclase activity induced by TNF-{alpha}, whereas MAb225 completely reversed EGF-induced sensitization of adenylyl cyclase (Fig. 8).


Figure 7
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  		Fig. 7. 10 µM forskolin-stimulated AC activity in cultured human airway smooth muscle cells following 48-h TNF-{alpha} (10 ng/ml, n = 6; A) or EGF (1 ng/ml, n = 4; B) treatment in the presence or absence of EGFR/erbB-2 inhibitor (inh) (10 µM for 30 min before each 48-h treatment). Within each experiment, values were determined in triplicate. Data represent means ± SE. *P < 0.05, ***P < 0.001 compared with control. ##P < 0.01 compared with TNF or EGF.

 

Figure 8
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  		Fig. 8. 10 µM forskolin-stimulated AC activity in cultured human airway smooth muscle cells following 48 h TNF-{alpha} (10 ng/ml) or EGF (1 ng/ml) treatment in the presence of monoclonal antibody against EGFR (MAb) or isotype control antibody IgG1 (2 µg/ml for 1 h before 48-h TNF-{alpha} or EGF treatment). Data represent means ± SE; n = 5. Within each experiment, values were determined in triplicate. ns, Not significant *P < 0.05, **P < 0.01 compared with control. ##P < 0.01 compared with EGF.

 
Effect of TNF-{alpha} on total protein expression of adenylyl cyclase isoforms. To determine whether an increased expression of adenylyl cyclase proteins could account for TNF-{alpha}-induced adenylyl cyclase sensitization, we performed immunoblot analysis of cultured human airway smooth muscle cells exposed to TNF-{alpha} for 48 h using an antibody against common sequence of all cloned mammalian membrane-associated adenylyl cyclase isoforms. There was no significant change in total adenylyl cyclase protein expression in cells left untreated vs. 48-h TNF-{alpha} treated (Fig. 9).


Figure 9
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  		Fig. 9. Western blot analysis of AC in cultured human airway smooth muscle cells using antibody against common sequence of nine AC isoforms. Cells were treated with (TNF) or without (cont) TNF-{alpha} (10 ng/ml) for 48 h. Membranes were subsequently reprobed with the antibody against beta-actin to confirm equal protein loading. Representative of five experiments is shown. Recombinantly expressed and purified human AC type VI (ACVI) from a baculovirus expression system was used as a positive control.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study illustrates for the first time in native human airway smooth muscle cells that a mechanism of TNF-{alpha}-induced adenylyl cyclase sensitization involves the transactivation of the EGFR and activation of raf-1 kinase. These studies link together two cellular signaling pathways not previously identified or linked in these cells: 1) TNF-{alpha}-induced transactivation of the EGFR with raf-1 kinase activation, and 2) raf-1 kinase-mediated sensitization of adenylyl cyclase.

Sensitization of adenylyl cyclase activity in human airway smooth muscle cells has previously been demonstrated in response to cytokines (9, 29), rhinovirus (9), and chronic activation of Gi-coupled receptors (7). Sensitization of adenylyl cyclase activity by chronic activation of Gi-coupled receptors was inhibited by PTX (7), consistent with a pathway shown in other cells Gi -> raf-1 kinase -> adenylyl cyclase; however, a role for raf-1 kinase in adenylyl cyclase sensitization in human airway smooth muscle cells has not been shown. In contrast, sensitization of adenylyl cyclase activity in response to IL-1beta was not PTX sensitive (9), in agreement with the present study using TNF-{alpha}.

Multiple previous studies have investigated the mechanisms of cytokine-induced G protein-coupled receptor (GPCR)-induced desensitization vs. adenylyl cyclase sensitization (9, 23, 29, 31). Pascaul et al. (29) demonstrated that concomitant treatment of human airway smooth muscle cells with IL-1beta and TNF-{alpha} or EGF for 18 h potentiated the induction of COX-2 and PGE2 and caused hyporesponsiveness of the GPCR for isoproterenol and prostaglandin E2. Conversely, 18-h treatment of human airway smooth muscle cells with TNF-{alpha}, IL-1beta, or EGF alone induced sensitization of adenylyl cyclase via a PGE2-independent pathway (29). However, the underlying mechanisms of TNF-{alpha}-induced sensitization of adenylyl cyclase were not well understood. Our present study agrees with these findings in that TNF-{alpha}-induced sensitization of forskolin-stimulated adenylyl cyclase activity was not affected by pretreatment with indomethacin and thus not dependent on prostaglandin synthesis. These previous studies have shown that cytokines typically decrease beta-adrenoceptor and prostaglandin receptor stimulation of adenylyl cyclase, while simultaneously increasing the direct (i.e., basal or forskolin stimulated) activity of adenylyl cyclase (9, 23, 29, 31). Thus the sensitization of adenylyl cyclase has been proposed to be a compensatory mechanism for the decreased ability of receptor pathways to stimulate adenylyl cyclase.

Sensitization of adenylyl cyclase has been most widely described after chronic stimulation of a Gi-protein coupled receptor. Classically following a chronic exposure to an agonist that acutely inhibits adenylyl cyclase activity, subsequent stimulation of adenylyl cyclase activity results in "overshoot" or supersensitization of adenylyl cyclase activity. This Gi protein-required sensitization of adenylyl cyclase is PTX sensitive. Therefore, in the present study, we questioned whether Gi proteins could be intermediates in the TNF-{alpha}-induced sensitization of adenylyl cyclase, but pretreatment with PTX failed to reverse the effect of TNF-{alpha}. Therefore, the cellular mechanisms of adenylyl cyclase sensitization by chronic Gi activation vs. TNF-{alpha} exposure are different.

Although the regulation of adenylyl cyclase activity is primarily by G protein or calcium-calmodulin interactions, alternate mechanisms involving regulation of the phosphorylation state of the adenylyl cyclase isoforms have also been identified. Because isoforms of adenylyl cyclases are known to be differentially regulated by multiple protein kinases, including PKA (33), PKC (11), raf-1 kinase (17, 36), and tyrosine kinases (32), we examined the effects of various protein kinase inhibitors. In the present study, we demonstrated that TNF-{alpha} treatment increased the phosphorylation of Ser338 on raf-1 kinase, which is indicative of enzyme activation (16). Raf-1 is the key protein kinase in the MAPK signal transduction cascade. Recently, it was shown that acute TNF-{alpha}-mediated enhancement of p42/44 MAPK phosphorylation in canine tracheal smooth muscle cells involved the activation of the Ras/Raf-1 pathway (16), consistent with the present study. In the present study, a MEK inhibitor failed to inhibit TNF-{alpha}-induced adenylyl cyclase sensitization, suggesting that raf-1-mediated sensitization of adenylyl cyclase does not require activation of the MAPK cascade. Although raf-1 is known to classically function upstream as an initiator of the Ras/Raf/ERK signaling cascade, raf-1 has been shown to directly activate adenylyl cyclase, independent of activation of the rest of the MAPK cascade (32), consistent with the present study.

We found that only GW5074, a selective raf-1 kinase inhibitor, could reverse the TNF-{alpha}-induced sensitization of adenylyl cyclase. Interestingly, addition of this inhibitor during the last 1 h or the last 6 h of the 48-h TNF exposure was sufficient to block the sensitization of adenylyl cyclase activity (Fig. 4). This would suggest that the raf-1 kinase-mediated activation of adenylyl cyclase is a reversible event and requires ongoing raf-1 kinase activation to maintain adenylyl cyclase in a phosphorylated and activated state.

In the present study, exposure to IL-1beta and EGF also sensitized adenylyl cyclase in a GW074-sensitive manner, suggesting that sensitization of adenylyl cyclase induced by these cytokines and growth factor may share a common signaling pathway involving raf-1 activation. Activation of EGFR is known to stimulate raf-1 and subsequently potentiate adenylyl cyclase type VI (AC6) activation in AC6-transfected human embryonic kidney-293 cells (5), which is consistent with the present study. Recently, TNF-{alpha} has been shown to induce EGFR transactivation, which, in turn, mediates cell proliferation and cell survival in various cell lines (2, 12). In the present study, an EGFR-selective inhibitor partially blocked TNF-{alpha}-induced sensitization of adenylyl cyclase and phosphorylation of raf-1 kinase. This suggests that transactivation of the EGFR is at least partly responsible for the effect of TNF-{alpha} on adenylyl cyclase sensitization. Transactivation of the EGFR is known to be induced by various stimuli, such as GPCR ligands, receptor tyrosine kinase agonists, cytokines, and cell adhesion elements (20). This involves both ligand-independent intracellular signaling mechanisms and metalloprotease-mediated releasing of the EGF-like ligands (20). Transactivation of the EGFR by TNF-{alpha}-induced metalloprotease processing of transforming growth factor-{alpha} has been demonstrated in hepatocytes and mammary epithelial cells (2, 12). Therefore, in the present study, we questioned whether certain EGF-like ligands could be intermediates in the TNF-{alpha}-induced sensitization of adenylyl cyclase. However, pretreatment with a MAb against the EGFR failed to reverse the effect of TNF-{alpha}, while completely blocking the effect of EGF on adenylyl cyclase activity. Taken together, these results suggest that a component of TNF-{alpha}-induced sensitization of adenylyl cyclase activity involves transactivation of the EGFR, leading to raf-1 kinase phosphorylation and activation, but that ligand occupancy of the EGFR is not required for raf-1 kinase activation in human airway smooth muscle cells.

Although PKC or tyrosine kinases are known to be upstream of raf-1 kinase and modulate its activation in some cells (14, 32), inhibitors of these kinases (GF109203X or genistein, respectively) failed to inhibit TNF-{alpha}-induced adenylyl cyclase sensitization. Moreover, the failure of inhibition of PKC, MEK, p38 MAPK, PI3 kinase, or tyrosine kinases inhibit TNF-{alpha}-induced adenylyl cyclase sensitization suggests that these kinases neither were directly responsible for altering adenylyl cyclase activity nor were these kinases acting upstream to activate raf-1 kinase. However, because the catalytic activity of raf-1 kinase is modulated by multiple independent mechanisms, it is possible that blocking an individual signaling pathway to raf-1 kinase may be insufficient to block its activation. For example, Varga et al. (36) reported that only simultaneous inhibition of both tyrosine kinase and PKC obliterated chronic opioid agonist-induced sensitization of adenylyl cyclase.

Recently, adenylyl cyclase II, V, VI, but not I, have been reported to be phosphorylated by Raf-1 (17) and directly sensitize adenylyl cyclase (32). We previously found that adenylyl cyclase V and AC6 are the major adenylyl cyclase isoforms expressed in cultured human airway smooth muscle cells (38). Thus the TNF-{alpha}-induced sensitization of adenylyl cyclase via Raf-1-dependent pathway likely involves these adenylyl cyclase isoforms. In the present study, treatment with TNF-{alpha} did not affect total protein expression of adenylyl cyclase isoforms, indicating that TNF-{alpha}-induced sensitization is mediated by activation, but not increased protein expression of adenylyl cyclase isoforms.

In summary, the present study demonstrates that chronic exposure to the inflammatory cytokine TNF-{alpha} induces Gi protein-independent sensitization of adenylyl cyclase in cultured human airway smooth muscle cells. This TNF-{alpha} effect is at least partly attributable to transactivation of the EGFR, which in turn activates and phosphorylates raf-1 kinase, which sensitizes adenylyl cyclase activity. A better understanding of the cellular signaling mechanisms underlying regulation of adenylyl cyclase activity under conditions of chronic cytokine exposure should improve therapies directed at airway smooth muscle tone in asthma.


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This work was supported by National Heart, Lung, and Blood Institute Grant HL-58519.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. W. Emala, Dept. of Anesthesiology, College of Physicians and Surgeons of Columbia Univ., 630 W. 168thSt. P&S Box 46, New York, New York 10032 (e-mail: cwe5{at}columbia.edu)

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


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  1. Ammit AJ, Hoffman RK, Amrani Y, Lazaar AL, Hay DW, Torphy TJ, Penn RB, Panettieri RA Jr. Tumor necrosis factor-alpha-induced secretion of RANTES and interleukin-6 from human airway smooth-muscle cells. Modulation by cyclic adenosine monophosphate. Am J Respir Cell Mol Biol 23: 794–802, 2000.[Abstract/Free Full Text]
  2. Argast GM, Campbell JS, Brooling JT, Fausto N. Epidermal growth factor receptor transactivation mediates tumor necrosis factor-induced hepatocyte replication. J Biol Chem 279: 34530–34536, 2004.[Abstract/Free Full Text]
  3. Babu KS, Davies DE, Holgate ST. Role of tumor necrosis factor alpha in asthma. Immunol Allergy Clin North Am 24: 583–597, 2004.[CrossRef][ISI][Medline]
  4. Baud V, Karin M. Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol 11: 372–377, 2001.[CrossRef][ISI][Medline]
  5. Beazely MA, Alan JK, Watts VJ. Protein kinase C and epidermal growth factor stimulation of Raf1 potentiates adenylyl cyclase type 6 activation in intact cells. Mol Pharmacol 67: 250–259, 2005.[Abstract/Free Full Text]
  6. Berry MA, Hargadon B, Shelley M, Parker D, Shaw DE, Green RH, Bradding P, Brightling CE, Wardlaw AJ, Pavord ID. Evidence of a role of tumor necrosis factor alpha in refractory asthma. N Engl J Med 354: 697–708, 2006.[Abstract/Free Full Text]
  7. Billington CK, Hall IP, Mundell SJ, Parent J, Panettieri RA, Benovic JL, Penn RB. Inflammatory and contractile agents sensitize specific adenylyl cyclase isoforms in human airway smooth muscle. Am J Respir Cell Mol Biol 21: 597–606, 1999.[Abstract/Free Full Text]
  8. Billington CK, Joseph SK, Swan C, Scott MG, Jobson TM, Hall IP. Modulation of human airway smooth muscle proliferation by type 3 phosphodiesterase inhibition. Am J Physiol Lung Cell Mol Physiol 276: L412–L419, 1999.[Abstract/Free Full Text]
  9. Billington CK, Pascual RM, Hawkins ML, Penn RB, Hall IP. Interleukin-1beta and rhinovirus sensitize adenylyl cyclase in human airway smooth-muscle cells. Am J Respir Cell Mol Biol 24: 633–639, 2001.[Abstract/Free Full Text]
  10. Broide DH, Lotz M, Cuomo AJ, Coburn DA, Federman EC, Wasserman SI. Cytokines in symptomatic asthmatic airways. J Allergy Clin Immunol 89: 958–967, 1992.[CrossRef][ISI][Medline]
  11. Chakrabarti S, Wang L, Tang WJ, Gintzler AR. Chronic morphine augments adenylyl cyclase phosphorylation: relevance to altered signaling during tolerance/dependence. Mol Pharmacol 54: 949–953, 1998.[Abstract/Free Full Text]
  12. Chen WN, Woodbury RL, Kathmann LE, Opresko LK, Zangar RC, Wiley HS, Thrall BD. Induced autocrine signaling through the epidermal growth factor receptor contributes to the response of mammary epithelial cells to tumor necrosis factor alpha. J Biol Chem 279: 18488–18496, 2004.[Abstract/Free Full Text]
  13. Chen Y, Liu J, Yu L. Functional coupling of a mu opioid receptor to G proteins and adenylyl cyclase: modulation by chronic morphine treatment. Addict Biol 1: 49–59, 1996.[CrossRef][ISI][Medline]
  14. Corbit KC, Trakul N, Eves EM, Diaz B, Marshall M, Rosner MR. Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein. J Biol Chem 278: 13061–13068, 2003.[Abstract/Free Full Text]
  15. Dhillon AS, Meikle S, Yazici Z, Eulitz M, Kolch W. Regulation of Raf-1 activation and signalling by dephosphorylation. EMBO J 21: 64–71, 2002.[CrossRef][ISI][Medline]
  16. Diaz B, Barnard D, Filson A, MacDonald S, King A, Marshall M. Phosphorylation of Raf-1 serine 338-serine 339 is an essential regulatory event for Ras-dependent activation and biological signaling. Mol Cell Biol 17: 4509–4516, 1997.[Abstract]
  17. Ding Q, Gros R, Gray ID, Taussig R, Ferguson SS, Feldman RD. Raf kinase activation of adenylyl cyclases: isoform-selective regulation. Mol Pharmacol 66: 921–928, 2004.[Abstract/Free Full Text]
  18. Doganci A, Sauer K, Karwot R, Finotto S. Pathological role of IL-6 in the experimental allergic bronchial asthma in mice. Clin Rev Allergy Immunol 28: 257–270, 2005.[CrossRef][ISI][Medline]
  19. Goncharova EA, Billington CK, Irani C, Vorotnikov AV, Tkachuk VA, Penn RB, Krymskaya VP, Panettieri RA Jr. Cyclic AMP-mobilizing agents and glucocorticoids modulate human smooth muscle cell migration. Am J Respir Cell Mol Biol 29: 19–27, 2003.[Abstract/Free Full Text]
  20. Gschwind A, Zwick E, Prenzel N, Leserer M, Ullrich A. Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transmission. Oncogene 20: 1594–1600, 2001.[CrossRef][ISI][Medline]
  21. Hotta K, Emala CW, Hirshman CA. TNF-alpha upregulates Gi{alpha} and Gq{alpha} protein expression and function in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 276: L405–L411, 1999.[Abstract/Free Full Text]
  22. Hsu HY, Twu YC. Tumor necrosis factor-alpha-mediated protein kinases in regulation of scavenger receptor and foam cell formation on macrophage. J Biol Chem 275: 41035–41048, 2000.[Abstract/Free Full Text]
  23. Laporte JD, Moore PE, Panettieri RA, Moeller W, Heyder J, Shore SA. Prostanoids mediate IL-1beta-induced beta-adrenergic hyporesponsiveness in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 275: L491–L501, 1998.[Abstract/Free Full Text]
  24. Lazaar AL, Panettieri RA Jr. Airway smooth muscle: a modulator of airway remodeling in asthma. J Allergy Clin Immunol 116: 488–495, 2005.[CrossRef][ISI][Medline]
  25. Lee JH, Johnson PR, Roth M, Hunt NH, Black JL. ERK activation and mitogenesis in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 280: L1019–L1029, 2001.[Abstract/Free Full Text]
  26. Mundell SJ, Olah ME, Panettieri RA, Benovic JL, Penn RB. Regulation of G protein-coupled receptor-adenylyl cyclase responsiveness in human airway smooth muscle by exogenous and autocrine adenosine. Am J Respir Cell Mol Biol 24: 155–163, 2001.[Abstract/Free Full Text]
  27. Ngoc PL, Gold DR, Tzianabos AO, Weiss ST, Celedon JC. Cytokines, allergy, asthma. Curr Opin Allergy Clin Immunol 5: 161–166, 2005.[ISI][Medline]
  28. Panettieri RA, Murray RK, DePalo LR, Yadvish PA, Kotlikoff MI. A human airway smooth muscle cell line that retains physiological responsiveness. Am J Physiol Cell Physiol 256: C329–C335, 1989.[Abstract/Free Full Text]
  29. Pascual RM, Billington CK, Hall IP, Panettieri RA Jr, Fish JE, Peters SP, Penn RB. Mechanisms of cytokine effects on G protein-coupled receptor-mediated signaling in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 281: L1425–L1435, 2001.[Abstract/Free Full Text]
  30. Salomon Y, Londos C, Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem 58: 541–548, 1974.[CrossRef][ISI][Medline]
  31. Shore SA, Laporte J, Hall IP, Hardy E, Panettieri RA Jr. Effect of IL-1 beta on responses of cultured human airway smooth muscle cells to bronchodilator agonists. Am J Respir Cell Mol Biol 16: 702–712, 1997.[Abstract]
  32. Tan CM, Kelvin DJ, Litchfield DW, Ferguson SS, Feldman RD. Tyrosine kinase-mediated serine phosphorylation of adenylyl cyclase. Biochemistry 40: 1702–1709, 2001.[CrossRef][Medline]
  33. Thomas JM, Hoffman BB. Chronic somatostatin treatment induces enhanced forskolin-stimulated cAMP accumulation in wild-type S49 mouse lymphoma cells but not in protein kinase-deficient mutants. Mol Pharmacol 35: 116–124, 1989.[Abstract]
  34. Tomlinson PR, Wilson JW, Stewart AG. Salbutamol inhibits the proliferation of human airway smooth muscle cells grown in culture: relationship to elevated cAMP levels. Biochem Pharmacol 49: 1809–1819, 1995.[CrossRef][ISI][Medline]
  35. Varga EV, Stropova D, Rubenzik M, Waite S, Roeske WR, Yamamura HI. Phosphorylation of adenylyl cyclase VI upon chronic delta-opioid receptor stimulation. Eur J Pharmacol 364: R1–R3, 1999.[CrossRef][ISI][Medline]
  36. Varga EV, Yamamura HI, Rubenzik MK, Stropova D, Navratilova E, Roeske WR. Molecular mechanisms of excitatory signaling upon chronic opioid agonist treatment. Life Sci 74: 299–311, 2003.[CrossRef][ISI][Medline]
  37. Watts VJ. Molecular mechanisms for heterologous sensitization of adenylate cyclase. J Pharmacol Exp Ther 302: 1–7, 2002.[Abstract/Free Full Text]
  38. Xu D, Isaacs C, Hall IP, Emala CW. Human airway smooth muscle expresses 7 isoforms of adenylyl cyclase: a dominant role for isoform V. Am J Physiol Lung Cell Mol Physiol 281: L832–L843, 2001.[Abstract/Free Full Text]




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