Effect of IL-1beta on CRE-dependent gene expression in human airway smooth muscle cells

Thomas Lahiri1, Paul E. Moore1, Simonetta Baraldo1, Timothy R. Whitehead1, Matthew D. McKenna1, Reynold A. Panettieri Jr.2, and Stephanie A. Shore1

1 Physiology Program, Harvard School of Public Health, Boston, Massachusetts 02115; and 2 Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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IL-1beta inhibits isoproterenol (ISO)-induced relaxation of cultured human airway smooth muscle (HASM) cells. The purpose of this study was to determine whether IL-1beta can also suppress ISO-induced cAMP response element (CRE)-dependent gene expression. ISO (10 µM) caused a marked increase in CRE-binding protein (CREB) phosphorylation, which was attenuated by IL-1beta (2 ng/ml). This effect of IL-1beta was abolished by the cyclooxygenase (COX) inhibitor indomethacin. To examine CRE-driven gene expression, we transiently transfected HASM cells with a construct containing CRE upstream of a luciferase reporter gene. ISO (6 h) caused a sixfold increase in luciferase activity. IL-1beta (24 h) alone also increased luciferase activity, although to a lesser extent (2-fold). However, the ability of ISO to elicit luciferase expression was markedly reduced in cells treated with IL-1beta . Indomethacin, the MEK and p38 inhibitors U-0126 and SB-203580, the protein kinase A inhibitor H-89, and dexamethasone each completely abolished the ability of IL-1beta to induce CRE-driven gene expression but only slightly increased the ability of ISO to induce CRE-driven gene expression in IL-1beta -treated cells. IL-1beta also attenuated dibutyryl cAMP-induced CRE-driven gene expression, but not dibutyryl cAMP-induced CREB phosphorylation. Tumor necrosis factor-alpha (10 ng/ml) also attenuated ISO-induced CRE-driven gene expression, even though it was without effect on ISO-induced cAMP formation or ISO-induced CREB phosphorylation. The results suggest that IL-1beta and tumor necrosis factor-alpha may attenuate the ability of beta -agonists to induce expression of genes with CRE in their regulatory regions at least in part through events downstream of CREB phosphorylation.

mitogen-activated protein kinase; cyclooxygenase-2; dexamethasone; cAMP response element binding; luciferase; tumor necrosis factor-alpha


    INTRODUCTION
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INTRODUCTION
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DECREASED RESPONSIVENESS to beta -adrenergic receptor agonists is a characteristic feature of asthma. Airways of asthmatic patients have a reduced capacity to dilate in response to beta -agonists in vivo (4) and in vitro (3). Animal models of asthma also exhibit decreased responses to beta -agonists (8). The mechanistic basis for these changes in responsiveness has not been established, but it is possible that cytokines released in the asthmatic airway may act directly on airway smooth muscle cells to exert these effects. For example, we previously reported that interleukin (IL)-1beta decreases the capacity of cultured human airway smooth muscle (HASM) cells to generate cAMP and to relax in response to isoproterenol (ISO) (31). Similar results have been obtained in other species (11, 35).

ISO exerts its effects in smooth muscle by binding to the beta 2-adrenergic receptor, which couples to the stimulatory G protein, Gs, which activates adenylyl cyclase, resulting in cAMP release. cAMP, in turn, activates protein kinase A (PKA), which results in cell relaxation through effects on K+ channels, Na+-K+-ATPases, Ca2+ sequestration, Ca2+ sensitivity of myosin, and inositol trisphosphate formation (28, 33). In addition to relaxing HASM cells, beta -agonist activation of PKA also induces changes in the transcription of genes that contain cAMP response elements (CRE) in their promoter regions (13, 27, 32). For example, the promoters of several genes that have been shown to be important for the regulation of smooth muscle cell function, such as the cyclooxygenase (COX)-2 gene (26), the IL-6 gene (1), and the gene for the beta 2-adrenergic receptor itself (19), contain CRE elements that are important in inducing gene transcription. Activation of CRE elements by ISO occurs through PKA phosphorylation of CRE-binding protein (CREB) at serine-133 (6, 29). Phosphorylation of CREB permits its interaction with CREB-binding protein (CBP) and p300, which interact with the basal transcriptional apparatus to initiate transcription (6).

The purpose of this study was to determine whether IL-1beta , in addition to its ability to inhibit beta -agonist-induced relaxation of smooth muscle, also inhibits beta -agonist-induced CRE-driven gene expression. To that end, we examined ISO-induced CREB phosphorylation in the presence and absence of IL-1beta . We also examined the effects of ISO and IL-1beta alone and in combination on luciferase expression in HASM cells transiently transfected with a luciferase reporter construct driven by multiple CRE elements. Our results indicate that IL-1beta substantially reduces the ability of ISO to evoke CRE-driven gene expression. Similar results were obtained with a representative gene, IL-6, which contains a CRE element in its promoter.

We previously reported that although IL-1beta decreases the ability of HASM cells to relax in response to ISO, IL-1beta does not affect HASM cell relaxation in response to dibutryl cAMP (DBcAMP), a cell-permeant analog of cAMP that directly activates PKA. IL-1beta also has no effect on the ability of forskolin, which directly activates adenylyl cyclase, to generate cAMP (31). These data support the hypothesis that the effect of IL-1beta on ISO-induced relaxation of HASM cells is mediated upstream of adenylyl cyclase, i.e., at the level of beta -receptor-Gs interactions. Our data also indicated that ISO-induced HASM relaxation occurs through a COX-2-dependent pathway (16), because we showed that IL-1beta markedly upregulates COX-2 expression and prostaglandin E2 (PGE2) release, that the effect of IL-1beta on ISO-induced cAMP formation is abolished in cells that are treated with COX-2 inhibitors, and that exogenous PGE2 mimics the effect of IL-1beta . Taken together, the data are consistent with the hypothesis that IL-1beta -induced PGE2 release results in increased basal cAMP formation and PKA activation, leading to phosphorylation of the beta 2-receptor, uncoupling it from Gs. Further investigations indicated that the effect of IL-1beta , which was dependent on activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase pathways acting to induce COX-2 expression (14, 15), could be blocked with glucocorticoids (20). For these reasons, we examined the effects of the COX inhibitor indomethacin, the MAP kinase kinase (MEK) inhibitor U-0126 (9), the p38 inhibitor SB-203580 (5), and the glucocorticoid dexamethasone on ISO-induced CRE-driven gene expression. We also examined the effect of IL-1beta on DBcAMP-induced CREB phosphorylation and CRE-driven gene expression. Because tumor necrosis factor-alpha (TNF-alpha ) is also present in asthmatic airways and can interact with IL-1beta to promote beta -adrenergic desensitization (18), we also examined the effect of TNF-alpha on ISO-induced CREB phosphorylation and CRE-driven gene expression.


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Cell culture. HASM cells were obtained from lung transplant donor tracheas in accordance with procedures approved by the University of Pennsylvania (Philadelphia) Committee on Studies Involving Human Beings. A segment of trachea just proximal to the carina was dissected under sterile conditions. Smooth muscle cells were isolated from the trachealis as previously described (21). For culture, the cells were plated into plastic flasks at 104 cells/cm2 in Ham's F-12 medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (0.1 mg/ml), amphotericin B (2.5 mg/ml), 12 mM NaOH, 1.6 µM CaCl2, 2 mM L-glutamine, and 25 mM HEPES. The medium was replaced every 3-4 days. The cells were passaged with 0.25% trypsin and 1 mM EDTA every 10-14 days. Confluent cells were deprived of serum and supplemented with insulin (5.7 µg/ml) and transferrin (5 µg/ml) 24 h before use. Cells in passages 4-7 from 18 different donors were used in the studies described below.

Western blotting for measurement of phosphorylated CREB. HASM cells were grown to confluence in six-well plates and deprived of serum for 24 h as described above. For measurement of phosphorylated CREB, cells were treated with ISO (10 µM for 15 min) and IL-1beta (2 ng/ml for 24 h) alone or in combination. Similar experiments were carried out in cells pretreated with the COX inhibitor indomethacin (1 µM) 2 h before treatment with IL-1beta . After treatment for the appropriate time, the culture medium was removed and the cells were washed with PBS and then lysed with 200 µl of extraction buffer [10 mM Tris · HCl buffer with 50 mM NaCl, 10 mM D-serine, 1 mM EDTA, 1 mM EGTA, 1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1 µg/ml pepstatin, and 10-2 U/ml aprotinin]. The cells were scraped from the plates, passed through a 25<FR><NU>5</NU><DE>8</DE></FR>-gauge needle, and solubilized by 10 s of sonication.

For CREB Western blots, supernatants of cell lysates were mixed with equal volumes of loading buffer [0.062 M Tris · HCl (pH 6.8), 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.01% (wt/vol) bromphenol blue] and boiled for 5 min. Solubilized proteins (30 µg/lane) were separated by SDS-polyacrylamide gel electrophoresis on 12% Tris-glycine gel (Invitrogen, Carlsbad, CA) under nonreducing conditions and transferred electrophoretically to a nitrocellulose membrane in transfer buffer (Pierce, Rockford, IL). The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature and probed with rabbit antiphosphorylated CREB (antiphosphorylated CREB; Cell Signaling Technologies, Beverly, MA) overnight at 4°C. The blots were then washed and incubated in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk with horseradish peroxidase-conjugated goat anti-rabbit IgG for 1 h. The proteins were visualized by light emission on film with enhanced chemiluminescent substrate (Cell Signaling). The bands visualized at 43 kDa for phosphorylated CREB were quantified with a laser densitometer. Band density values are expressed as arbitrary optical density units. Cell extracts were also used to examine the expression of nonphosphorylated CREB by Western blotting (Cell Signaling).

Transfection of HASM cells. After passage, HASM cells were grown in complete medium for 72 h (60-80% confluence) in six-well plates. Before transfection, the medium was changed to 1% fetal bovine serum. HASM cells were transfected with 0.5 µg of pCRE-luciferase (pCRE-Luc), designed for monitoring the CRE signal transduction pathway (Clontech, Palo Alto, CA), and 0.5 µg of a beta -galactosidase control vector to normalize for differences in transfection efficiency from well to well. The pCRE-Luc vector contains three copies of a consensus CRE-binding sequence fused to a TATA-like promoter region upstream of the gene for firefly luciferase. Further details about the vector are available on the manufacturer's website. The CRE and beta -galactosidase vectors were cotransfected with Fugene 6 (Roche, Indianapolis, IN) according to the manufacturer's protocol. At 24 h after transfection, the medium was replaced with serum-free medium containing insulin and transferrin as described above. The cells were then incubated with IL-1beta (2 ng/ml for 24 h) and ISO (10 µM for 6 h) alone or in combination. The above protocol was also repeated in the presence or absence of the MEK inhibitor U-0126 (10 µM; Promega, Madison, WI). Inhibition of MEK prevents the phosphorylation and activation of ERK. We also repeated the protocol in the presence and absence of the p38 inhibitor SB-203580 (3 µM; Calbiochem-Novabiochem, La Jolla, CA), indomethacin (1 µM), or dexamethasone (1 µM). Each of these drugs was administered 2 h before addition of IL-1beta . We also repeated the protocol using DBcAMP (1 mM) instead of ISO and using TNF-alpha (10 ng/ml) instead of IL-1beta . The cells were lysed with reporter lysis buffer (Promega) and harvested. The samples were assayed for luciferase activity by scintillation counting and for beta -galactosidase activity by spectrophotometry with the beta -galactosidase enzyme assay system (Promega). The results of the experiments are reported as mean luciferase activity normalized to beta -galactosidase activity. Drugs and reagents were obtained from Sigma (St. Louis, MO) unless otherwise indicated.

We also examined the effect of IL-1beta (2 ng/ml for 24 h) and ISO (10 µM for 6 h) alone or in combination on luciferase activity in cells transfected with an IL-6 promoter luciferase reporter construct (pIL-6-Luc 651) or an identical construct mutated at the nuclear factor-kappa B (NF-kappa B) site 63 bp upstream of the transcription start site (pIL-6-Luc 651 Delta NF-kappa B) (2, 7). These constructs were the kind gift of Dr. Oliver Eickelberg (Yale University, New Haven, CT) with permission from Dr. Shigeru Katamine (Nagasaki University, Nagasaki, Japan) (7).

Measurement of cAMP. Cells were treated with TNF-alpha (10 ng/ml) or left untreated. After 20 h, cells were harvested by brief exposure to trypsin and EDTA, resuspended in serum-free medium with or without TNF-alpha , and plated at 105 cells/well in 24-well plates. After the cells were allowed to readhere for 4 h at 37°C, the medium was replaced with 0.5 ml of PBS containing 0.1 mM IBMX (to prevent degradation of cAMP by phosphodiesterases) and 300 µM ascorbic acid (to prevent oxidation of ISO). After 30 min, ISO (10 µM) or diluent was added to the cells. Cells were incubated for an additional 10 min and then removed from the incubator and placed on ice. Ice-cold ethanol (1 ml) was added to lyse the cells. The lysate was centrifuged at 2,000 g for 15 min at 4°C, and the supernatant was removed, evaporated to dryness, and stored at -70°C until assay. On the day of assay, the cell lysate was resuspended in 200-500 µl of assay buffer. Some samples were diluted to ensure that they fell within the limits of the standard curve. cAMP was assayed using a Rainen cAMP 125I radioimmunoassay kit (New England Nuclear).

Statistics. The significance of drug treatment on CREB phosphorylation (densitometry), CRE-driven luciferase expression, and cAMP formation was assessed by ANOVA using drug treatment and experimental day as main effects. Follow-up t-tests were used to assess where the drug treatment effect lay. Bonferroni's rule was used to correct for multiple comparisons. P < 0.05 was considered significant.


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ISO-induced CREB phosphorylation. Figure 1A shows the effect of ISO (10 µM for 15 min) and IL-1beta (2 ng/ml for 24 h) on CREB phosphorylation. ISO caused a large increase in CREB phosphorylation compared with control cells. When cells were treated with IL-1beta (2 ng/ml) 24 h before addition of ISO, ISO-induced CREB phosphorylation was largely suppressed (Fig. 1A). Similar results were obtained in cells from three HASM cell donors, and densitometric analysis of the data indicated that the decrease in ISO-induced CREB phosphorylation caused by IL-1beta was statistically significant (P < 0.05; Fig. 1B). In contrast, IL-1beta did not attenuate ISO-induced CREB phosphorylation in cells that had been treated with indomethacin (1 µM) 2 h before addition of IL-1beta (Fig. 1).


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Fig. 1.   A: Western blot showing phosphorylated cAMP response element (CRE)-binding protein (phospho-CREB) expression in response to isoproterenol (ISO, for 15 min) and/or IL-1beta (for 24 h). Experiments were performed in the presence or absence of indomethacin (Indo). ST, phospho-CREB standard. B: laser densitometric quantification, expressed as relative optical density units, of phospho-CREB expression in human airway smooth muscle (HASM) cells from 3 donors. *P < 0.05 compared with ISO alone.

ISO-induced CRE-driven gene expression. HASM cells were transfected with a pCRE-Luc construct and treated with ISO (10 µM for 6 h) and IL-1beta (2 ng/ml for 24 h) alone and in combination. ANOVA indicated a statistically significant effect of drug treatment (P < 0.001) on CRE-driven luciferase expression (Fig. 2). ISO caused an approximately sixfold increase (P < 0.001) in luciferase expression normalized for beta -galactosidase expression compared with untreated cells, whereas IL-1beta resulted in an approximately twofold increase in luciferase expression (P < 0.001). When cells were treated first with IL-1beta and 18 h later with ISO, the ability of ISO to increase luciferase activity was markedly diminished (P < 0.001). Indeed, the effect of ISO (difference between ISO-treated and control cells) in IL-1beta -treated cells averaged only 12 ± 3% of the response to ISO in cells not treated with IL-1beta .


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Fig. 2.   Luciferase expression (normalized for beta -galactosidase expression) in response to ISO (10 µM for 6 h) and/or IL-1beta (2 ng/ml for 24 h). Values are means ± SE from 34-36 HASM cell wells in each case and were obtained on 18 experimental days in cells from 12 donors. *P < 0.001 compared with ISO alone.

To examine the mechanistic basis for the effects of IL-1beta on CRE-driven gene expression, HASM cells were treated with the COX inhibitor indomethacin (1 µM), with U-0126 (10 µM), an inhibitor of the enzyme MEK that phosphorylates ERK, with the p38 inhibitor SB-203580, with the glucocorticoid dexamethasone (1 µM), or with the PKA inhibitor H-89 (1 µM). In each case, cells were treated with the inhibitor 2 h before treatment with IL-1beta (2 ng/ml for 24 h). None of these treatments had any effect on basal luciferase expression measured in control untreated cells (data not shown). However, each of these treatments significantly reduced IL-1beta -induced CRE-mediated gene expression to control values (Fig. 3), suggesting that this effect of IL-1beta may be mediated through induction of COX-2.


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Fig. 3.   Effect of cyclooxygenase-2 (COX-2) inhibitor Indo (1 µM for 26 h), MEK inhibitor U-0126 (10 µM for 26 h), p38 inhibitor SB-203580 (3 µM for 26 h), dexamethasone (Dex, 1 µM for 26 h), and protein kinase A inhibitor H-89 (1 µM for 26 h) on IL-1beta (2 ng/ml for 24 h)-induced changes in CRE-driven luciferase expression normalized for beta -galactosidase expression. Values are means ± SE of data from 6-12 HASM cell wells in each case and were obtained on 3-5 experimental days in cells from 3-5 donors. *P < 0.001 compared with control.

We also examined the effect of indomethacin, U-0126, SB-203580, and dexamethasone on ISO-induced CRE-driven gene expression in cells with and without IL-1beta (2 ng/ml for 24 h) treatment. The PKA inhibitor H-89 was not examined, because at the concentrations that inhibit PKA, H-89 has been shown to have activity as a beta -adrenergic receptor antagonist (23), precluding our ability to use it in experiments involving effects of ISO. In cells not treated with IL-1beta , dexamethasone induced an ~40% increase in ISO-induced CRE-driven gene expression (P < 0.01), but U-0126, SB-203580, and indomethacin were without effect (data not shown). As described above, the overall effect of ISO (10 µM for 6 h, difference between ISO-treated and control cells) in IL-1beta -treated cells averaged only 12 ± 3% of the response to ISO in cells not treated with IL-1beta . Each of the inhibitors, except SB-203580, caused a small but significant increase in the response to ISO in IL-1beta -treated cells (Fig. 4), but even in the presence of the inhibitors, the response to ISO was still much less than the response obtained in cells not treated with IL-1beta (100%). For example, even in the presence of indomethacin, which caused the most substantial increase in the response to ISO, the ISO-induced luciferase expression in IL-1beta -treated cells averaged only ~40% of that obtained in cells not treated with IL-1beta .


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Fig. 4.   Effect of COX-2 inhibitor Indo (1 µM for 26 h), MEK inhibitor U-0126 (10 µM for 26 h), p38 inhibitor SB-203580 (3 µM for 26 h), and Dex (1 µM for 26 h) on IL-1beta (2 ng/ml for 24 h)-induced changes in ISO (10 µM, 6 h)-induced CRE-driven luciferase expression. Results are expressed as effect of ISO (luciferase expression induced in the presence of ISO minus luciferase expression induced in the absence of ISO) in IL-1beta treated cells as a percentage of the effect of ISO in cells not treated with IL-1beta . Values are means ± SE from 6-12 HASM cell wells in each case and were obtained on 3-5 experimental days in cells from 3-5 donors. *P < 0.05 compared with control.

DBcAMP-induced CREB phosphorylation and CRE-driven gene expression. DBcAMP (1 mM for 15 min), like ISO, increased CREB phosphorylation (Fig. 5). However, in contrast to the effect of IL-1beta on ISO-induced CREB phosphorylation (Fig. 1), IL-1beta (2 ng/ml for 24 h) did not alter DBcAMP-induced CREB phosphorylation (Fig. 5). Nevertheless, IL-1beta did reduce DBcAMP-induced CRE-dependent gene expression (Fig. 6). DBcAMP caused a marked increase in luciferase expression in untreated cells transfected with a pCRE-Luc reporter. However, in cells treated with IL-1beta , the effect of DBcAMP was significantly reduced (P < 0.05). Taken together, the results suggest that the effects of IL-1beta on DBcAMP-induced changes in CRE-driven gene expression must lie downstream of CREB phosphorylation.


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Fig. 5.   A: Western blot showing phospho-CREB expression in response to dibutyryl cAMP (DBcAMP, 10-3 M for 15 min) and/or TNF-alpha (10 ng/ml for 24 h). B: densitometric analysis of phospho-CREB Western blots in cells from 4 HASM donors. Values are means ± SE.



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Fig. 6.   CRE-driven luciferase expression in response to DBcAMP (10-3 M for 6 h) and/or IL-1beta (2 ng/ml for 24 h). Values are means ± SE from 3 HASM cell donors studied in duplicate. *P < 0.05 compared with DBcAMP alone.

Effect of TNF-alpha on ISO-induced CREB phosphorylation and CRE-driven gene expression. We previously reported that TNF-alpha has only minimal effects on the ability of ISO to relax HASM cells (18). To confirm that TNF-alpha also does not alter ISO-induced changes in cAMP formation, as has previously been reported (22), we measured changes in cAMP induced by ISO in cells treated with or without TNF-alpha (10 ng/ml for 24 h). In contrast to IL-1beta , which increases basal cAMP formation (31), TNF-alpha did not alter basal cAMP formation in HASM cells (43.3 ± 6.8 vs. 47.6 ± 7.1 pmol/106 cells). ISO (10 µM for 15 min) increased cAMP formation, but there was no significant difference in the response to ISO in cells treated with TNF-alpha compared with control cells (209.5 ± 22.6 and 208.5 ± 23.7 pmol/106 cells, respectively). Similarly, TNF-alpha did not alter ISO-induced CREB phosphorylation (Fig. 7). In contrast to IL-1beta , TNF-alpha did not alter basal CRE-driven gene expression in HASM cells (Fig. 8), likely because TNF-alpha does not induce COX-2 expression in HASM cells (18). However, similar to IL-1beta , TNF-alpha did attenuate the ability of ISO to induce luciferase expression (Fig. 8). Taken together, these results suggest that the effect of TNF-alpha on CRE-driven gene expression must lie downstream of CREB phosphorylation.


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Fig. 7.   Western blot showing phospho-CREB expression in response to ISO (10 µM for 15 min) and/or TNF-alpha (10 ng/ml for 24 h). Similar results were obtained in cells from 3 donors.



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Fig. 8.   CRE-driven luciferase expression in response to ISO (10 µM for 6 h) and/or TNF-alpha (10 ng/ml for 24 h). Values are means ± SE of data from 6-12 HASM cell wells in each case and were obtained on 3 experimental days in cells from 2 donors. *P < 0.05 vs. ISO alone.

Effect of IL-1beta on ISO-induced IL-6 promoter activity. To confirm the relevance of our results for genes with CRE in their promoters, we repeated these experiments in cells transfected with pIL-6-Luc 651 instead of the CRE-Luc construct (Fig. 9). ISO stimulates IL-6 promoter activity in HASM cells, and this effect of ISO is dependent on the presence of a CRE at position -154 (2). Our results (Fig. 9A) indicated that ISO caused an 84 ± 34% increase (P < 0.05) in luciferase in pIL-6-Luc 651-transfected cells but failed to increase IL-6 promoter activity in IL-1beta -treated cells. We also repeated the experiments in cells transfected with pIL-6-Luc 651 Delta NF-kappa B (Fig. 9B) to eliminate effects of IL-1beta on IL-6 promoter activity that were independent of its effects on the beta -adrenergic pathway. Mutation of the NF-kappa B binding site at position -63 of the IL-6 promoter completely abolished the ability of IL-1beta to stimulate IL-6 promoter activity. ISO still caused a robust increase in promoter activity in control cells but failed to increase promoter activity in cells treated with IL-1beta . Taken together, the results indicate that IL-1beta also attenuates ISO-induced CRE-driven IL-6 expression.


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Fig. 9.   pIL-6-Luc 651 activity (A) and pIL-6-Luc 651 Delta NF-kappa B activity (B) in response to ISO (10 µM for 6 h) and/or IL-1beta (2 ng/ml for 24 h). Values are means ± SE of data from 3-6 HASM cell wells in each case and were obtained in cells from 3 donors. *P < 0.05 compared with cells in the same treatment group without ISO. Results were normalized for beta -galactosidase activity and are expressed relative to control cells.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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We previously reported that IL-1beta suppresses ISO-induced cAMP formation and relaxation in HASM cells (31). The results of the present study demonstrate that IL-1beta also inhibits the ability of ISO to induce CREB phosphorylation and CRE-driven gene expression (Figs. 1 and 2). The effect of IL-1beta on ISO-induced CREB phosphorylation appears to require COX-2. In contrast, the effect of IL-1beta on ISO-induced CRE-driven gene expression is only slightly attenuated by COX-2 inhibitors and appears to result, at least in part, from effects downstream of CREB phosphorylation, because IL-1beta also suppressed DBcAMP-driven gene expression (Fig. 6) without altering DBcAMP-induced CREB phosphorylation (Fig. 5).

We previously reported that although IL-1beta causes a marked inhibition of the ability of ISO to induce cAMP formation and relax airway smooth muscle, the cytokine has no effect on responses to DBcAMP or forskolin (31). These results suggest that the effect of IL-1beta on ISO-induced cAMP formation lies upstream of adenylyl cyclase. Because beta 2-receptor expression and Gs expression were also unaffected, we reasoned that IL-1beta was acting to uncouple the beta -adrenergic receptor from Gs (31). We subsequently demonstrated that the ability of ISO to cause cell relaxation and to induce cAMP formation was restored by COX-2 inhibitors and mimicked by PGE2 (16). Similar results have been obtained by other investigators (22). Because IL-1beta by itself caused a small but significant increase in cAMP formation, we reasoned that IL-1beta might be acting by increasing COX-2 expression and PGE2 release, resulting in increased cAMP formation. We further reasoned that the increased PKA activity resulting from this increase in cAMP could be causing the decrease in beta -adrenergic responsiveness by phosphorylating the beta -receptor (12, 17, 25), uncoupling it from Gs.

Our results indicate that this mechanism also accounts for the ability of IL-1beta to attenuate ISO-induced CREB phosphorylation, because this effect was abolished by indomethacin (Fig. 1). In contrast, this mechanism is likely to account for only a small part of the effect of IL-1beta on ISO-induced CRE-driven gene expression, because the COX inhibitor indomethacin restored only a small portion of the ability of ISO to induce CRE-driven luciferase expression (Fig. 4), likely as a result of its effects on CREB phosphorylation. Furthermore, U-0126 and SB-203580, inhibitors of the ERK and p38 MAP kinase pathways, and dexamethasone also reversed only a very small part of the effect of IL-1beta on ISO-induced CRE-driven gene expression (Fig. 4), even though all these compounds substantially attenuate IL-1beta -induced COX-2 expression and completely abolish the loss of ISO-induced relaxation responses that are observed in IL-1beta -treated cells (14, 15, 20). It is unlikely that the inability of these agents to restore the effects of ISO on CRE-driven gene expression is due to a lack of efficacy, because we previously reported that, at the concentrations used, U-0126 virtually abolishes the ability of IL-1beta to induce ERK phosphorylation (14), whereas indomethacin, dexamethasone, and SB-203580 also abolish the ability of IL-1beta to induce PGE2 release in HASM cells (15, 16, 20). Instead, the results suggest that an additional mechanism accounts for the majority of the effect of IL-1beta on ISO-induced CRE-driven gene expression.

We do not know the precise details of this additional mechanism. However, the observation that IL-1beta does not alter DBcAMP-induced CREB phosphorylation (Fig. 5) but does attenuate CRE-driven gene expression mediated by DBcAMP (Fig. 6) suggests that there must be effects of IL-1beta on events downstream of CREB phosphorylation. Because interaction of CBP and p300 with phosphorylated CREB is vital to the formation of the basal transcriptional apparatus that allows transcription of CRE-dependent genes (6), it is possible that the ability of IL-1beta to attenuate ISO- or DBcAMP-induced CRE-driven gene expression is the result of effects on recruitment of CBP to phosphorylated CREB. CBP interacts not only with CREB, but also with other transcription factors (6, 10, 29, 30), some of which, e.g., NF-kappa B and AP-1, are activated by IL-1beta in HASM cells (20), and it has been hypothesized that nuclear competition between transcription factors for limiting amounts of CBP results in mutual repression of the ability of these factors to activate transcription (30). Alternatively, it has also been reported that the C/EBP family of transcription factors can interact with CRE in some genes (24). We have reported that IL-1beta induces C/EBPbeta expression in HASM cells (34), and it is possible that this transcription factor competes with CREB for binding to CRE.

Although IL-1beta attenuated ISO-induced CREB phosphorylation (Fig. 1), our results suggest that this effect played only a minor role in the reduction in ISO-induced CRE-driven gene expression that was caused by IL-1beta . First, IL-1beta also attenuated DBcAMP-induced CRE-driven gene expression, but this occurred without any effect on DBcAMP-induced CREB phosphorylation (Figs. 5 and 6). Second, indomethacin completely restored the ability of ISO to induce CREB phosphorylation in IL-1beta -treated cells (Fig. 1), but even after restoration of CREB phosphorylation, IL-1beta continued to attenuate ISO-induced CRE-driven gene expression (Fig. 4), albeit not to the same extent as in cells not treated with indomethacin.

TNF-alpha , like IL-1beta , is increased in asthmatic airways. Our results indicate that TNF-alpha , like IL-1beta , also suppresses CRE-driven gene expression in HASM cells (Fig. 8). Because TNF-alpha did not alter ISO-induced cAMP formation, it is likely that the cytokine must be exerting its effects downstream of beta -receptor-Gs coupling and adenylyl cyclase activation. Because TNF-alpha does not alter cell stiffness responses to DBcAMP (18), the cytokine cannot be affecting PKA activity. Taken together with our results indicating no effect of TNF-alpha on ISO-induced CREB phosphorylation (Fig. 7), the data indicate that the effect of TNF-alpha on CRE-driven gene expression (Fig. 8) is also mediated downstream of CREB.

IL-1beta induction of COX-2 appears to be only minimally involved in the effect of IL-1beta on ISO-induced CRE-driven gene expression (Fig. 4). However, COX-2 does appear to contribute to the ability of IL-1beta alone to increase CRE-driven gene expression in HASM cells, because the COX inhibitor indomethacin completely blocked this effect (Fig. 3). In addition, U-0126 and SB-203580 inhibited the effects of IL-1beta on CRE-driven gene expression, consistent with the requirement of the p38 and ERK MAP kinases for IL-1beta -induced COX-2 expression (14, 15). Dexamethasone, which blocks IL-1beta -induced COX-2 expression (20), was also effective in abolishing IL-1beta -induced CRE-driven gene expression. Hence, it is likely that IL-1beta induces CRE-driven gene expression as follows. IL-1beta induces COX-2, leading to PGE2 release. PGE2 acts on EP receptors on the smooth muscle cells to induce cAMP formation and subsequent PKA induction. PKA then phosphorylates CREB, which recruits CBP, leading to gene expression. These results are consistent with our previous observations that IL-1beta causes a small but significant increase in basal cAMP formation in HASM cells that can be abolished by COX-2 inhibitors (16) and that the PKA inhibitor H-89 inhibited the effects of IL-1beta on CRE-driven gene expression (Fig. 3).

In summary, our results indicate that ISO-induced CRE-driven gene expression is suppressed by pretreatment with IL-1beta or TNF-alpha in HASM cells and that the effect of these cytokines is likely to be at least partly mediated by events downstream of CREB phosphorylation. These effects of IL-1beta may have important implications for the expression of genes such as IL-6, which contain CRE in their regulatory regions. Thus IL-1beta , in addition to inhibiting beta -agonist-induced HASM cell relaxation, also inhibits the effect of beta -agonists on CRE-dependent gene expression, both of which may be important in asthma.


    ACKNOWLEDGEMENTS

The authors thank I. Schwartzman and T. Church for excellent technical assistance.


    FOOTNOTES

This work was supported by National Institutes of Health Grants HL-67664, HL-56383, HL-33009, HL-55301, HL-64063, and AI-40203. P. E. Moore was supported by National Heart, Lung, and Blood Institute Grant HL-04395 and grants from the American Lung Association and the Charles H. Hood Foundation.

Address for reprint requests and other correspondence: S. A. Shore, Physiology Program, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115 (E-mail: sshore{at}hsph.harvard.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.

July 26, 2002;10.1152/ajplung.00231.2001

Received 18 June 2001; accepted in final form 22 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ammit, AJ, Hoffman RK, Amrani Y, Lazaar AL, Hay DW, Torphy TJ, Penn RB, and 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.   Ammit, AJ, Lazaar AL, Irani C, O'Neill GM, Gordon ND, Amrani Y, Penn RB, and Panettieri RA, Jr. Tumor necrosis factor-alpha -induced secretion of RANTES and interleukin-6 from human airway smooth muscle cells: modulation by glucocorticoids and beta -agonists. Am J Respir Cell Mol Biol 26: 465-474, 2002[Abstract/Free Full Text].

3.   Bai, TR. Abnormalities in airway smooth muscle in fatal asthma. Am Rev Respir Dis 141: 552-557, 1990[ISI][Medline].

4.   Barnes, PJ, and Pride NB. Dose-response curves to inhaled beta -adrenoceptor agonists in normal and asthmatic subjects. Br J Clin Pharmacol 15: 677-682, 1983[ISI][Medline].

5.   Cuenda, A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR, and Lee JC. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 364: 229-233, 1995[ISI][Medline].

6.   De Cesare, D, Fimia GM, and Sassone-Corsi P. Signaling routes to CREM and CREB: plasticity in transcriptional activation. Trends Biochem Sci 24: 281-285, 1999[ISI][Medline].

7.   Eickelberg, O, Pansky A, Mussmann R, Bihl M, Tamm M, Hildebrand P, Perruchoud AP, and Roth M. Transforming growth factor-beta 1 induces interleukin-6 expression via activating protein-1 consisting of JunD homodimers in primary human lung fibroblasts. J Biol Chem 274: 12933-12938, 1999[Abstract/Free Full Text].

8.   Emala, C, Black C, Curry C, Levine MA, and Hirshman CA. Impaired beta -adrenergic receptor activation of adenylyl cyclase in airway smooth muscle in the basenji-greyhound dog model of airway hyperresponsiveness. Am J Respir Cell Mol Biol 8: 668-675, 1993[ISI][Medline].

9.   Favata, MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, and Trzaskos JM. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273: 18623-18632, 1998[Abstract/Free Full Text].

10.   Gerritsen, ME, Williams AJ, Neish AS, Moore S, Shi Y, and Collins T. CREB-binding protein/p300 are transcriptional coactivators of p65. Proc Natl Acad Sci USA 94: 2927-2932, 1997[Abstract/Free Full Text].

11.   Hakonarson, H, Herrick DJ, Serrano PG, and Grunstein MM. Mechanism of cytokine-induced modulation of beta -adrenoceptor responsiveness in airway smooth muscle. J Clin Invest 97: 2593-2600, 1996[Abstract/Free Full Text].

12.   Hausdorff, WP, Bouvier M, O'Dowd BF, Irons GP, Caron MG, and Lefkowitz RJ. Phosphorylation sites on two domains of the beta 2-adrenergic receptor are involved in distinct pathways of receptor desensitization. J Biol Chem 264: 12657-12665, 1989[Abstract/Free Full Text].

13.   Lalli, E, Lee JS, Lamas M, Tamai K, Zazopoulos E, Nantel F, Penna L, Foulkes NS, and Sassone-Corsi P. The nuclear response to cAMP: role of transcription factor CREM. Philos Trans R Soc Lond B Biol Sci 351: 201-209, 1996[ISI][Medline].

14.   Laporte, JD, Moore PE, Abraham JH, Maksym GN, Fabry B, Panettieri RA, Jr, and Shore SA. Role of ERK MAP kinases in responses of cultured human airway smooth muscle cells to IL-1beta . Am J Physiol Lung Cell Mol Physiol 277: L943-L951, 1999[Abstract/Free Full Text].

15.   Laporte, JD, Moore PE, Lahiri T, Schwartzman IN, Panettieri RA, Jr, and Shore SA. p38 MAP kinase regulates IL-1beta responses in cultured airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 279: L932-L941, 2000[Abstract/Free Full Text].

16.   Laporte, JD, Moore PE, Panettieri RA, Moeller W, Heyder J, and 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].

17.   Lohse, MJ, Benovic JL, Caron MG, and Lefkowitz RJ. Multiple pathways of rapid beta 2-adrenergic receptor desensitization. Delineation with specific inhibitors. J Biol Chem 265: 3202-3211, 1990[Abstract/Free Full Text].

18.   Moore, PE, Lahiri T, Laporte JD, Church T, Panettieri RA, Jr, and Shore SA. Synergism between TNF-alpha and IL-1beta in airway smooth muscle cells: implications for beta -adrenergic responsiveness. J Appl Physiol 91: 1467-1474, 2001[Abstract/Free Full Text].

19.   Moore, PE, Lahiri T, Silverman ES, Panettieri RA, and Shore SA. Transcriptional regulation of the beta 2-adrenergic receptor in human airway smooth muscle cells (Abstract). Am J Respir Crit Care Med 163: A778, 2001.

20.   Moore, PE, Laporte JD, Gonzalez S, Moller W, Heyder J, Panettieri RA, Jr, and Shore SA. Glucocorticoids ablate IL-1beta -induced beta -adrenergic hyporesponsiveness in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 277: L932-L942, 1999[Abstract/Free Full Text].

21.   Panettieri, RA, Murray RK, DePalo LR, Yadvish PA, and 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].

22.   Pang, L, Holland E, and Knox AJ. Role of cyclo-oxygenase-2 induction in interleukin-1beta -induced attenuation of cultured human airway smooth muscle cell cyclic AMP generation in response to isoprenaline. Br J Pharmacol 125: 1320-1328, 1998[Abstract].

23.   Penn, RB, Parent JL, Pronin AN, Panettieri RA, Jr, and Benovic JL. Pharmacological inhibition of protein kinases in intact cells: antagonism of beta -adrenergic receptor ligand binding by H-89 reveals limitations of usefulness. J Pharmacol Exp Ther 288: 428-437, 1999[Abstract/Free Full Text].

24.   Potter, S, Mitchell MD, Hansen WR, and Marvin KW. NF-IL6 and CRE elements principally account for both basal and interleukin-1beta -induced transcriptional activity of the proximal 528 bp of the PGHS-2 promoter in amnion-derived AV3 cells: evidence for involvement of C/EBPbeta . Mol Hum Reprod 6: 771-778, 2000[Abstract/Free Full Text].

25.   Premont, RT, Inglese J, and Lefkowitz RJ. Protein kinases that phosphorylate activated G protein-coupled receptors. FASEB J 9: 175-182, 1995[Abstract/Free Full Text].

26.   Rikitake, Y, Hirata K, Kawashima S, Takeuchi S, Shimokawa Y, Kojima Y, Inoue N, and Yokoyama M. Signaling mechanism underlying COX-2 induction by lysophosphatidylcholine. Biochem Biophys Res Commun 281: 1291-1297, 2001[ISI][Medline].

27.   Sassone-Corsi, P. Coupling gene expression to cAMP signalling: role of CREB and CREM. Int J Biochem Cell Biol 30: 27-38, 1998[ISI][Medline].

28.   Scheid, CR, Honeyman TW, and Fay FS. Mechanism of beta -adrenergic relaxation of smooth muscle. Nature 277: 32-36, 1979[ISI][Medline].

29.   Shaywitz, AJ, and Greenberg ME. CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu Rev Biochem 68: 821-861, 1999[ISI][Medline].

30.   Sheppard, KA, Phelps KM, Williams AJ, Thanos D, Glass CK, Rosenfeld MG, Gerritsen ME, and Collins T. Nuclear integration of glucocorticoid receptor and nuclear factor-kappa B signaling by CREB-binding protein and steroid receptor coactivator-1. J Biol Chem 273: 29291-29294, 1998[Abstract/Free Full Text].

31.   Shore, SA, Laporte J, Hall IP, Hardy E, and Panettieri RA, Jr. Effect of IL-1beta on responses of cultured human airway smooth muscle cells to bronchodilator agonists. Am J Respir Cell Mol Biol 16: 702-712, 1997[Abstract].

32.   Tamai, KT, Monaco L, Nantel F, Zazopoulos E, and Sassone-Corsi P. Coupling signalling pathways to transcriptional control: nuclear factors responsive to cAMP. Recent Prog Horm Res 52: 121-139, 1997[ISI][Medline].

33.   Torphy, TJ. beta -Adrenoceptors, cAMP and airway smooth muscle relaxation: challenges to the dogma. Trends Pharmacol Sci 15: 370-374, 1994[ISI][Medline].

34.   Whitehead, TR, Moore PE, McKenna MD, Silverman ES, Panettieri RA, Jr, and Shore SA. Involvement of C/EBPbeta in oncostatin-M- and IL-1beta -induced COX-2 expression in human airway smooth muscle (HASM) cells (Abstract). Am J Respir Crit Care Med 165: A116, 2002.

35.   Wills-Karp, M, Uchida Y, Lee JY, Jinot J, Hirata A, and Hirata F. Organ culture with proinflammatory cytokines reproduces impairment of the beta -adrenoceptor-mediated relaxation in tracheas of a guinea pig antigen model. Am J Respir Cell Mol Biol 8: 153-159, 1993[ISI][Medline].


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