Cyclooxygenase-2 and MicroRNA-155 Expression Are Elevated in Asthmatic Airway Smooth Muscle Cells

Abstract

Cyclooxygenase-2 (COX-2) expression and PGE₂ secretion from human airway smooth muscle cells (hASMCs) may contribute to β₂-adrenoceptor hyporesponsiveness, a clinical feature observed in some patients with asthma. hASMCs from patients with asthma exhibit elevated expression of cytokine-responsive genes, and in some instances this is attributable to an altered histone code and/or microRNA expression. We hypothesized that COX-2 expression and PGE₂ secretion might be elevated in asthmatic hASMCs in response to proinflammatory signals in part due to altered histone acetylation and/or microRNA expression. hASMCs obtained from nonasthmatic and asthmatic human subjects were treated with cytomix (IL-1β, TNF-α, and IFN-γ). A greater elevation of COX-2 mRNA, COX-2 protein, and PGE₂ secretion was observed in the asthmatic cells. We investigated histone H3/H4-acetylation, transcription factor binding, mRNA stability, p38 mitogen-activated protein kinase signaling, and microRNA (miR)-155 expression as potential mechanisms responsible for the differential elevation of COX-2 expression. We found that histone H3/H4-acetylation and transcription factor binding to the COX-2 promoter were similar in both groups, and histone H3/H4-acetylation did not increase after cytomix treatment. Cytomix treatment elevated NF-κB and RNA polymerase II binding to similar levels in both groups. COX-2 mRNA stability was increased in asthmatic cells. MiR-155 expression was higher in cytomix-treated asthmatic cells, and we show it enhances COX-2 expression and PGE₂ secretion in asthmatic and nonasthmatic hASMCs. Thus, miR-155 expression positively correlates with COX-2 expression in the asthmatic hASMCs and may contribute to the elevated expression observed in these cells. These findings may explain, at least in part, β₂-adrenoceptor hyporesponsiveness in patients with asthma.

Clinical Relevance

This research finds that asthmatic airway smooth muscle exhibits enhanced expression of cyclooxygenase-2 (COX-2), which may contribute to β₂-adrenoceptor desensitization in asthma. Furthermore, the authors identify microRNA (miR)-155, which enhances COX-2 expression, as being up-regulated in asthmatic airway smooth muscle and possibly responsible for the elevated COX-2 expression observed in these cells. This manuscript highlights the role miR-155 may play in promoting β₂-adrenoceptor desensitization and highlights miR as a potential treatment target in asthma.

Hyporesponsiveness to β₂-adrenoceptor agonists due to heterologous desensitization is an important limitation to the successful control of asthma symptoms (1–5). Several of the cytokines that are elevated in the bronchoalveolar lavage fluid of patients with asthma (including IL-1β and TNF-α) promote β₂-adrenoceptor hyporesponsiveness (1, 3–7). In vitro studies of airway smooth muscle suggest that cytokine-mediated up-regulation of cyclooxygenase-2 (COX-2) leads to enhanced secretion of PGE₂, activation of the cAMP/PKA pathway, and disruption of coupling of β₂-adrenoceptors with Gαs (2, 4, 5). Cytokine exposure of human airway smooth muscle cells (hASMCs) from patients with asthma induces a much greater expression of several cytokine targets, including eotaxin-1, CXCL10, and CXCL8, than is observed in nonasthmatic hASMCs (8–10). COX-2 expression is also cytokine responsive in hASMCs, suggesting that it may be increased in asthma due to an inflammatory milieu that alters COX-2 transcription, translation, or both. The level of COX-2 expression in the airway smooth muscle of patients with asthma is not well defined. Early studies in primary hASMCs showed that stimulation with bradykinin resulted in reduced COX-2 expression in asthmatic hASMCs compared with nonasthmatics hASMCs (11). As a consequence, asthmatic hASMCs also secreted less PGE₂ when treated with bradykinin (11). In a separate study, hASMCs from patients with asthma secreted similar levels of PGE₂ when treated with IL-1β and/or TNF-α, but COX-2 expression was not reported (12). Asthmatic airway smooth muscle exhibits increased surface expression of E-prostanoid receptors 2 and 3, indicating the cells may be hypersensitive to autocrine PGE₂ signaling (13). Other studies show an up-regulation of COX-2 and its reaction products in asthma (14, 15). Epigenetic regulation of COX-2 expression is an emerging area of interest, and controller mechanisms include microRNAs (e.g., microRNA [miR]-155) and histone posttranslation modifications (e.g., acetylation) (16–19). Indeed, reduced histone acetylation and miR-146a expression have been linked to alterations in COX-2 expression in idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease, respectively (17, 20). These regulatory mechanisms have been demonstrated to be altered in asthmatic hASMCs (21, 22) but not in the context of COX-2 expression. On this basis we hypothesized that asthmatic hASMCs would express greater amounts of cytokine-induced COX-2 and secrete higher levels of PGE₂ than cells from nonasthmatic subjects because histone acetylation and/or miRNA regulation of COX-2 expression is altered in the asthmatic cells. Specifically, we first investigated whether asthmatic hASMCs express more COX-2 and secrete more PGE₂ than nonasthmatic hASMCs when treated with a complex mixture of cytokines (IL-1β, TNF-α, and IFN-γ) that mimics inflammation in asthmatic airways. Second, we tested whether the difference in the magnitude of COX-2 induction was due to a modification in the “histone code,” altered expression of microRNAs (miRNAs or miRs) that regulate COX-2 expression, or a combination of both.

Materials and Methods

Cell Culture

Primary asthmatic and nonasthmatic hASMCs were isolated from nontransplantable donor lung tissue or from resected lung tissue by enzymatic digestion at the University of Chicago or University of Manitoba, respectively. All tissue procurement and cell culture studies were conducted following protocols approved by the Human Research Ethics Board (University of Manitoba) and Institutional Review Boards for Protection of Human Subjects of the University of Chicago and the University of South Alabama. Donor characteristics are listed in Table 1. Cells cultured in 5% CO₂ at 37°C in DMEM supplemented with 5% FBS, 0.5 μg/L basic fibroblast growth factor, 2 μg/L epidermal growth factor, 50 U/ml penicillin, and 100 μg/ml streptomycin. Cells growth-arrested for experiments in DMEM/Ham’s F-12 supplemented with 0.25 μg/ml insulin, 0.11 μg/ml transferrin, and 0.1 ng/ml selenium. Nonasthmatic and asthmatic hASMCs were assayed at passages 6 and 7. Cells were treated with cytomix (10 ng/ml IL-1β, 10 ng/ml TNF-α, and 10 ng/ml IFN-γ), treated with individual cytokines, or left untreated for the indicated time periods. Media and supplements were obtained from Life Technologies (Grand Island, NY), Cell Generation (Fort Collins, CO), Miltenyi Biotec (Bergisch Gladbach, Germany), or Sigma-Aldrich (St. Louis, MO).

Table 1.

Characteristics of Asthmatic and Nonasthmatic Human Airway Smooth Muscle Cell Lung Donors from the University of Chicago

Asthma Status	Age (yr)	Gender	Race or Ethnicity	Cause of Death	Asthma Therapy
Asthmatic
	12	Male	Hispanic	Car accident	Yes
	16	Male	Black	Unknown	Unknown
	21	Male	Back	Head Trauma	Yes
	34	Male	White	Unknown	Unknown
	47	Female	White	Unknown	Unknown
	51	Female	Black	Intracranial hemorrhage	Yes
	57	Male	Hispanic	Cardiovascular event	Yes
Mean age, yr	34
SEM	7
Nonasthmatic
	27	Male	White	Head trauma	n/a
	29	Female	White	Head trauma	n/a
	42	Male	Black	Unknown	n/a
	44	Female	White	Intracranial hemorrhage	n/a
	77	Female	White	Intracranial hemorrhage	n/a
Mean age, yr	44
SEM	9

Definition of abbreviation: n/a, not applicable.

Donor characteristics were not obtained for nonasthmatic human airway smooth muscle cells from University of Manitoba, and these cells were only used in experiments investigating microRNA-155 function.

Western Blotting

Protein samples were prepared as previously described (23). For COX-2 and mitogen-activated protein kinases (MAPKs), 10 to 15 μg and 25 μg of protein, respectively, were separated by SDS-PAGE and transferred to nitrocellulose. Equal loading was verified using FastBlot stain (G-biosciences, St. Louis, MO), and the region around 43 kD is presented. Antibodies for COX-2 (sc-1745), p38 MAPK (sc-535), and phospho-p38 MAPK (9216) were obtained from Santa Cruz Biotechnology (Dallas, TX) or Cell Signaling (Beverly, MA). Fluorescent secondary antibodies (IRDye; LI-COR, Lincoln, NE) were detected with a near-infrared laser scanner (Odyssey; LI-COR). Integrated intensities were expressed relative to a COX-2–positive control sample, total MAPK, or control mimic COX-2 as appropriate.

PGE₂ Assay

Further details are provided in the online supplement.

RNA Isolation, Quantitative RT-PCR, and mRNA Stability

Total RNA was purified using silica-based columns after extraction with phenol/guanidine. RNA quality and concentration were assessed by spectrophotometry. Reverse transcription was performed using a cDNA synthesis kit (iScript; Bio-Rad, Hercules, CA), and real-time amplification was performed using RT² SYBR Green qPCR mastermixes (Qiagen, Valencia, CA) with previously published primers for COX-2 and 18S rRNA (23). COX-2 mRNA abundance relative to abundance of 18S rRNA was calculated using the 2^−ΔΔCt method (24). COX-2 mRNA stability was assayed as previously described using quantitative RT-PCR (qRT-PCR) and 5 μg/ml actinomycin D (23). MiR-155–5p abundance was assayed by Taqman qRT-PCR and quantified using a miRNA standard curve.

MicroRNA Mimic and Inhibitor Transfection

Details are provided in the online supplement.

Chromatin Immunoprecipitation

Methods were adapted from those published by Knox and colleagues (16, 20, 25). Details are provided in the online supplement.

Statistical Analyses

Statistical analyses were performed using GraphPad Prism (Version 4; GraphPad, San Diego, CA). Selected pairs of treatments were compared using one-way ANOVA with Bonferroni’s test. MiR-155 expression within either nonasthmatic or asthmatic groups was compared with a defined “control” using a one-way ANOVA and Dunnett’s test. COX-2 mRNA half-lives rate constants were compared using an unpaired Student’s t test.

Results

To compare COX-2 expression in asthmatic and nonasthmatic cells, hASMCs in culture were treated with individual cytokines or cytomix for 20 hours (26). Previously a time-course study was performed to investigate COX-2 expression in hASMCs treated with cytomix, and it was demonstrated that COX-2 expression peaks at 20 hours (23). Consistent with previous reports (23, 27), treatment for 20 hours with IL-1β alone or with cytomix increased COX-2 protein abundance, with COX-2 abundance being greater in asthmatic than in nonasthmatic cells (Figure 1). COX-2 protein abundance did not differ between untreated asthmatic and nonasthmatic cells or when cultures were treated with individual cytokines. Enhanced accumulation of COX-2 in asthmatic cells was only evident when the cultures were exposed to a combination of cytokines. To determine if increased COX-2 protein abundance correlated with changes in mRNA abundance, we measured COX-2 mRNA steady-state levels by qRT-PCR. Asthmatic and nonasthmatic cells were grown with and without cytomix for 20 hours. In the absence of cytomix, COX-2 mRNA abundance was very low and similar in all cultures (Figure 2A), which is consistent with our assessment of protein abundance (Figure 1). Cytomix treatment increased COX-2 mRNA in asthmatic and nonasthmatic cells as previously reported (23) but, as we observed for protein, COX-2 mRNA abundance was higher in asthmatic compared with nonasthmatic cells (Figure 2A). These results suggest differential levels of COX-2 transcriptional activation and/or transcript stability in asthmatic versus nonasthmatic hASMCs. The predominant prostaglandin that is synthesized by hASMCs is PGE₂, with others (PGI₂, PGD₂, PGF_2α) secreted at lower levels (27). To test whether elevated levels of COX-2 mRNA and protein result in enhanced PGE₂ secretion from asthmatic cells, we treated cultures with cytomix (0.1, 1, or 10 ng/ml each of IL-1β, TNF-α, and IFN-γ) for 20 hours. Cytomix increased the secretion of PGE₂ from nonasthmatic (Figure 2B) and asthmatic cells (Figure 2C), consistent with a previous report (24). PGE₂ secretion from asthmatic cells (Figure 2C) was greater than from nonasthmatic cells (Figure 2B) at the highest concentration of cytomix used. We also tested the impact of treating cytomix-exposed cells with the COX-2 selective inhibitor NS-398 or pretreatment with dexamethasone (27, 28) and found that PGE₂ secretion was completely inhibited (Figures 2B and 2C). This demonstrates that PGE₂ secretion requires COX-2 activity and is blocked by corticosteroid treatment in asthmatic and nonasthmatic hASMCs.

Figure 1.

Cyclooxygenase-2 (COX-2) protein abundance is greater in asthmatic human airway smooth muscle cells (hASMCs) treated with cytomix than in nonasthmatic cells. COX-2 protein abundance was compared in nonasthmatic and asthmatic hASMCs that were left untreated or were treated with cytomix (10 ng/ml each of IL-1β, TNF-α, and IFN-γ) or with the individual cytokines for 20 hours. COX-2 protein abundance was assayed by Western blotting, and COX-2–integrated intensity was normalized to a COX-2–positive control (PC) sample that was loaded on each gel. (A) Representative blots showing COX-2 immunoreactivity and stained total protein (region corresponding to actin). (B) Normalized COX-2–integrated intensity: nonasthmatic (open bars) and asthmatic (solid bars). Protein data (mean ± SEM) obtained from four or five nonasthmatic and four to six asthmatic hASMC donors (n = 8–12). *P < 0.001 versus nonasthmatic 20-hour untreated cells. ^†P < 0.001 versus asthmatic 20-hour untreated cells. ^‡P < 0.001 versus nonasthmatic cells treated with cytomix. All P values were calculated using one-way ANOVA with Bonferroni’s post hoc testing.

Figure 2.

COX-2 mRNA abundance and PGE₂ secretion are greater in asthmatic hASMCs treated with cytomix than in nonasthmatic hASMCs. hASMCs from nonasthmatic (open bars) and asthmatic (solid bars) hASMCs were left untreated or were treated with cytomix (10 ng/ml each of IL-1β, TNF-α, and IFN-γ) for 20 hours. (A) COX-2 mRNA abundance was determined by quantitative RT-PCR using the 2^−ΔΔCt method and expressed relative to cytomix-treated nonasthmatic cells. Data (mean ± SEM) were obtained from four or five nonasthmatic and five to seven asthmatic hASMC donors (n = 8–14). PGE₂ secretion was compared in nonasthmatic (B) and asthmatic (C) cells that were treated with cytomix (0.1–10 ng/ml of each cytokine) for 20 hours. For the 10 ng/ml dose, additional cells were pretreated with 10 μM dexamethasone or 10 μM NS-398. PGE₂ secreted into cell culture medium was assayed by enzyme immunoassay, and values were normalized relative to cell number. Dotted lines represent mean PGE₂ concentration present in the media of untreated cells at 20-hour PGE₂ data (mean ± SEM) obtained from five nonasthmatic and five asthmatic hASMC donors for dose response (n = 10) and from three nonasthmatic and four asthmatic hASMCs donors for NS-398/dexamethasone treatments (n = 6–8). *P < 0.001 versus nonasthmatic treated with cytomix. ^§P < 0.05 versus nonasthmatic cells treated for 20 hours with 10 ng/ml cytomix. All P values were calculated using one-way ANOVA with Bonferroni’s post hoc testing.

Evidence exists correlating differential histone acetylation and transcription factor binding in asthmatic cells with differential gene expression (10, 21). To define the molecular mechanisms responsible for the differential COX-2 expression that we observed, we tested for altered histone H3/H4 acetylation and transcription factor binding at the COX-2 promoter (−299 to +6 bp relative to the transcription start site) in the asthmatic cells. Chromatin immunoprecipitation assays were designed to measure peak acetylation and transcription factor binding as based on results from Knox and colleagues (16), and were performed after treatment of asthmatic and nonasthmatic hASMCs with or without cytomix treatment for 30 minutes. The relative amounts of histone H3 and H4 acetylation and the occupancy of the COX-2 promoter by RNA polymerase II, NF-κB, and C/EBP-β were assessed. After cytomix treatment, there was no increase in histone H3 or H4 acetylation at the COX-2 promoter compared with the untreated time controls (Figures 3A and 3B). We also used chromatin immunoprecipitation assays to investigate transcription factors and RNA polymerase II binding within the COX-2 promoter and RNA polymerase II binding within the COX-2 (PTGS2) gene. Binding of RNA polymerase II within the COX-2 gene was assayed at +1,727 to +2,093 bp relative to the transcription start site, a region that partially overlaps exon 4. We found that cytomix treatment resulted in increased binding of NF-κB (Figure 3C) and RNA polymerase II (Figure 3E) at the COX-2 promoter and of RNA polymerase II (Figure 3F) within the COX-2 gene to the same extent in nonasthmatic and asthmatic hASMCs. In contrast, C/EBP-β binding at the COX-2 promoter was refractory to cytomix treatment in all hASMC cultures (Figure 3D). Our observations that binding of NF-κB and RNA polymerase II to the COX-2 gene are similar in asthmatic and nonasthmatic cells strongly indicate that COX-2 gene transcription is not altered in asthmatic hASMCs. These results furthermore suggest that increased COX-2 protein is more likely due to posttranscriptional regulation.

Figure 3.

Histone H3/H4 pan-acetylation, transcription factor, and RNA polymerase II binding to the COX-2 promoter/gene is similar in nonasthmatic and asthmatic hASMCs. (A) Histone H3 pan-acetyl, (B) H4 pan-acetyl, (C) NF-κB p65, (D) C/EBP-β, (E) RNA polymerase II immunoprecipitated within COX-2 promoter, and (F) RNA polymerase II immunoprecipitated within the COX-2 gene. Asthmatic (solid bars) and nonasthmatic (open bars) hASMCs were treated with cytomix (10 ng/ml each of IL-1β, TNF-α, and IFN-γ) or were left untreated for 30 minutes. Data are expressed relative to input DNA. Dotted line = negative control average % input. The negative control dotted line is not visible for RNA polymerase II bound to the COX-2 promoter (E). Data (mean ± SEM) obtained from four or five nonasthmatic and five asthmatic hASMC donors (n = 4–5). *P < 0.01 versus untreated nonasthmatic cells. ^†P < 0.05 versus untreated asthmatic cells. ^‡P < 0.001 versus untreated nonasthmatic cells. ^§P < 0.001 versus untreated asthmatic cells. All P values were calculated using one-way ANOVA with Bonferroni’s post hoc testing.

Because regulation of mRNA stability can be an important regulatory mechanism for controlling protein abundance, we compared COX-2 mRNA stability in nonasthmatic and asthmatic hASMCs treated with cytomix. The half-life of COX-2 mRNA was measured in hASMCs after cessation of cytomix treatment at 20 hours. In the presence of actinomycin D, which blocks further transcription, COX-2 mRNA half-life was found to be significantly longer in asthmatic hASMCs compared with their nonasthmatic counterparts (Figure 4). Because the activation of the p38 MAPK pathway by cytomix is known to increase the stability of COX-2 mRNA in hASMCs (23), we also compared p38 MAPK phosphorylation kinetics in nonasthmatic and asthmatic hASMCs treated with cytomix. We found that the level of p38 MAPK phosphorylation induced after 15 minutes was lower in asthmatic hASMCs compared with the nonasthmatic cells (Figure 5); we saw no statistically significant differences at other time points. This demonstrates that enhanced expression and mRNA stability for COX-2 in asthmatic hASMCs is not likely due to increased activation of pathways involving p38 MAPK.

Figure 4.

COX-2 mRNA stability is elevated in asthmatic hASMCs treated with cytomix. Nonasthmatic (open boxes/dashed line) and asthmatic (solid boxes/solid line) hASMCs were treated with cytomix (10 ng/ml each of IL-1β, TNF-α, and IFN-γ) for 20 hours followed by removal of cytomix and by incubation with 5 μg/ml actinomycin D (Act. D) for up to 2 hours. RNA was extracted at 0, 15, 30, 60, and 120 minutes after Act. D addition. COX-2 mRNA abundance was quantified by quantitative RT-PCR using the 2^−ΔΔCt method and expressed relative to 0-minute mRNA levels. Inset shows results calculated by nonlinear regression and includes rate constant (k, per minute) and half-life (t_1/2, min) for decay to the plateau. Data (mean ± SEM) obtained from two nonasthmatic and two asthmatic hASMC donors in quadruplicate (n = 8). *P = 0.016 versus rate constant for nonasthmatic hASMCs calculated using an unpaired Student’s t test.

Figure 5.

p38 mitogen-activated protein kinase (MAPK) phosphorylation is compromised in asthmatic hASMCs treated with cytomix. Nonasthmatic (open boxes/dashed line) and asthmatic (solid boxes/solid line) hASMCs were treated with cytomix (10 ng/ml each of IL-1β, TNF-α, and IFN-γ) for 0, 5, 15, or 30 minutes, and proteins were assayed by Western blotting. Phosphorylated p38 abundance is expressed relative to total p38 MAPK. Representative blots are displayed below. Data (mean ± SEM) obtained from four nonasthmatic and five asthmatic hASMCs (n = 8–10). *P < 0.01 versus 15-minute nonasthmatic cells calculated using one-way ANOVA with Bonferroni’s post hoc testing.

Because our data appear to rule out any influence of histone H3/H4 acetylation status, transcription factor binding, or p38 MAPK activation on differential COX-2 gene expression in asthmatic hASMCs, we next investigated whether microRNAs may regulate COX-2 protein abundance. Although several microRNAs can theoretically bind the 3′ untranslated region and regulate the stability of COX-2 transcripts, we focused on miR-155 because it has been associated with proinflammatory effects that promote cytokine-responsive gene expression in several lung cell types (18, 29). MiR-155 abundance was assayed after cytokine or cytomix stimulation, and levels were compared between nonasthmatic and asthmatic hASMCs. MiR-155 abundance increased in hASMCs treated with IL-1β alone, TNF-α alone, or cytomix (Figure 6). IFN-γ alone did not significantly increase miR-155 levels. All treatments that significantly increased miR-155 abundance did so to an even greater extent in asthmatic hASMCs (Figure 6). The cytokine-mediated induction of miR-155 observed here is consistent with results obtained in other cell types (30, 31). To assess a direct functional role for miR-155 in COX-2 expression, we next examined whether a miR-155 oligonucleotide mimic was sufficient to enhance COX-2 protein abundance and PGE₂ secretion in nonasthmatic hASMCs to levels similar to those observed in the asthmatic cells. When nonasthmatic cells were transfected with miR-155 mimics (30 nM) and treated with cytomix, we observed greater COX-2 protein abundance compared with cells transfected with a control miRNA mimic cel-miR-67 (Figure 7A). The maximum effect of the miR-155 mimic on COX-2 protein abundance was observed at 30 nM (see Figure E1 in the online supplement). In addition, we also observed greater PGE₂ secretion in nonasthmatic cells transfected with miR-155 mimic, as compared with cells transfected with control miRNA mimic (Figure 7B). The miR-155 mimic potentiated cytomix-stimulated COX-2 expression in nonasthmatic cells to levels greater than those observed in asthmatic cells (Figure 1). These results show that delivery of a miR-155 mimic to hASMCs is sufficient to modulate a nonasthmatic cell to an asthmatic phenotype. We next investigated the impact of silencing miR-155 by transfecting nonasthmatic and asthmatic hASMCs with a miR-155 inhibitor and assessed the cell response to cytomix exposure. We observed a modest but significant reduction in COX-2 protein abundance in the nonasthmatic cells transfected with 20 nM of the miR-155 inhibitor (Figure 7C). We observed a similar reduction in COX-2 protein abundance in asthmatic cells when compared with nonasthmatic cells transfected with up to 80 nM miR-155 inhibitor (Figure 7C). Treatment of asthmatic cells with 40 and 80 nM of the miR-155 inhibitor reduced PGE₂ secretion 27 and 28%, respectively (Figure E2). These results show that increased expression of miR-155 is sufficient and required to enhance the abundance of COX-2 protein, which increases PGE₂ secretion. Furthermore, we demonstrate that inhibition of miR-155 function in asthmatic hASMCs is sufficient to reduce COX-2 protein abundance and PGE₂ secretion.

Figure 6.

MicroRNA (miR)-155 abundance is greater in asthmatic than in nonasthmatic hASMCs treated with cytomix. MiR-155 abundance in nonasthmatic (open bars) and asthmatic (solid bars) hASMCs treated with cytomix (10 ng/ml each of IL-1β, TNF-α, and IFN-γ) or with the individual cocktail components or in hASMCs left untreated for 20 hours. Data (mean ± SEM) obtained from five nonasthmatic and seven asthmatic hASMCs (n = 10–14). ^†P < 0.05 versus asthmatic 20-hour untreated cells calculated using one-way ANOVA with Dunnett’s post hoc testing. *P < 0.01 versus nonasthmatic or asthmatic 20-hour untreated cells calculated using one-way ANOVA with Dunnett’s post hoc testing. ^‡P < 0.01 versus nonasthmatic cells treated with cytomix calculated using one-way ANOVA with Bonferroni’s post hoc testing.

Figure 7.

miR-155 enhances COX-2 expression and PGE₂ secretion in hASMCs treated with cytomix. HASMCs were transfected with 30 nM control (open bars) or miR-155 (solid bars) mimics and then treated with or without cytomix (10 ng/ml each of IL-1β, TNF-α, IFN-γ) for 20 hours. (A) COX-2 protein abundance normalized to cytomix-treated controls with representative blot (lanes in duplicate). (B) PGE₂ secretion was normalized to cytomix-treated controls. Nonasthmatic hASMCs were transfected with 20 nM control (open bars) or miR-155 (solid bars) inhibitor, and asthmatic hASMCs were transfected with 20 to 80 nM control (open bars) or miR-155 (solid bars) inhibitor and treated as above. (C) COX-2 protein abundance normalized to cytomix-treated controls with representative blots (lanes in duplicate). Data (mean ± SEM) obtained from three to five hASMC donors (n = 6–10). *P < 0.001 versus cytomix-treated control (Cont.) mimic or inhibitor (Inh.). ^†P < 0.01 versus cytomix-treated control inhibitor. P values calculated using one-way ANOVA with Bonferroni’s post hoc testing.

Discussion

β₂-adrenoceptor hyporesponsiveness has been observed in some patients with asthma, and this has been linked to cytokine-induced COX-2 expression and the resulting autocrine effects of secreted PGE₂ (1–5). Because asthmatic hASMCs show enhanced cytokine-responsive gene expression, we hypothesized that COX-2 expression would be elevated by cytokine stimulation in asthmatic hASMCs. Our results support this hypothesis and show that cytomix stimulation yields greater abundance of COX-2 protein and mRNA, which is associated with increased secretion of PGE₂. If this were to occur in airway smooth muscle of patients with asthma, greater localized synthesis of PGE₂ could result and contribute to heterologous desensitization and β₂-adrenoceptor hyporesponsiveness (1–5). Elevated expression of cytokine-responsive genes is an important feature of the secretory activity of airway smooth muscle cells and contributes to the inflammatory milieu in asthmatic airways (32). It has been known for some time that the proinflammatory phenotype of asthmatics persists in culture, and recent work is uncovering mechanisms for this feature. Molecular mechanisms responsible for elevated cytokine-responsive gene expression in asthmatic hASMCs include histone methylation, histone acetylation, microRNA expression, and altered extracellular matrix profiles (8, 21, 22, 25). Recent understanding of epigenetic mechanisms of gene expression suggest that among these processes, histone posttranslational modifications may be particularly important in establishing a persistent inflammatory phenotype (33). Our studies addressed this and revealed no differences in histone acetylation associated with the COX-2 gene between the nonasthmatic and asthmatic hASMCs. Furthermore, we observed no change in histone acetylation in nonasthmatic or asthmatic cells after treating them with cytomix despite results from Knox and colleagues, indicating that IL-1β alone is sufficient to induce changes in histone acetylation at the COX-2 promoter in hASMCs (16). The lack of a change in histone acetylation may be because we included IFN-γ in the cytokine cocktail. IFN-γ can inhibit histone acetyltransferase activity and enhance the activation of histone deacetylases (34). Collectively, our data and already published data may indicate that although histone acetylation can be dynamic under some inflammatory conditions, it is not likely the sole determinant of enhanced COX-2 expression in asthmatic hASMCs. A limitation of assessing global changes in histone modifications is that changes in acetylation at a limited number of sites may enhance binding of transcription factors to the COX-2 promoter without a detectable change in overall acetylation levels. Increased binding of transcription factors to a cytokine-responsive gene promoter has been observed in asthmatic hASMCs, and this correlated with enhanced expression of CXCL8 in asthmatic cells (10). It is plausible that a similar phenomenon might occur at the COX-2 promoter in asthmatic hASMCs, so we investigated NF-κB, C/EBP-β, and RNA polymerase II binding to the COX-2 promoter. Our results do not support a correlation between increased transcription factor binding and increased COX-2 expression in asthmatic cells. Our findings differ from those of Knox and colleagues (10, 21) in which elevated histone acetylation and transcription factor binding at the CXCL8 promoter were observed in asthmatic hASMCs and correlated with hypersecretion of CXCL8 from asthmatic hASMCs. Our results do not exclude the possibility that histone posttranslational modifications alter gene expression in asthmatic hASMCs but show that no fundamental differences in RNA polymerase II, NF-κB, or C/EBP-β binding to the COX-2 promoter are present in the cells. Therefore, we investigated other posttranscriptional processes to explain the enhanced COX-2 expression. Posttranscriptional regulation of COX-2 expression includes the regulation of mRNA stability through a complex interaction of A/U-rich elements with RNA binding proteins that stabilize, destabilize, or inhibit translation of bound transcripts (35). The function of many RNA binding proteins is regulated by phosphorylation, commonly mediated by the p38 MAPK pathway, which upon activation stabilizes select mRNAs that contain A/U-rich elements in their 3′-untranslated region, including COX-2 (23, 36–39). A role for this pathway seemed plausible because we observed increased COX-2 stability in hASMCs after 20 hour stimulation with cytokines (Figure 4), which is a novel observation in asthmatic hASMCs. Although COX-2 message was stabilized, it does not appear to be due to enhanced activation of the p38 MAPK pathway. p38 MAPK phosphorylation (an index of activation) was reduced in the asthmatic hASMCs. Although p38 MAPK is an important regulator of COX-2 mRNA stability, it is probably not the sole determinant. Recent studies in fibroblasts, chondrocytes, and cancer cells clearly show miRNA-mediated regulation of COX-2 transcript stability (17, 19, 40).

We then investigated a role for miR-155 in regulating COX-2 protein abundance because miR-155 promotes cytokine responsive gene expression in other cell types, including enhancement of COX-2 expression in RAW264.7 cells (18, 30, 41). Furthermore, studies in miR-155 knockout mice have demonstrated that the miRNA promotes allergic airway inflammation and mucus hypersecretion in asthma (42). MiR-155 expression increases in a time-dependent manner in retinal epithelial cells treated with cytomix similar to what was observed by Singer and colleagues (24) for COX-2 expression in hASMCs, although COX-2 expression plateaus at 20 hours (32). We demonstrate that in hASMCs, cytomix increased miR-155 levels and COX-2 protein abundance after 20 hours of stimulation. A miR-155 gain-of-function approach increased COX-2 protein abundance and PGE₂ secretion in nonasthmatic cells. MiR-155 loss of function yielded a modest reduction of COX-2 protein in nonasthmatic and asthmatic cells. Furthermore, miR-155 inhibition reduced PGE₂ secretion in asthmatic cells. Altogether, the results are consistent with miR-155 enhancing COX-2 expression in asthmatic hASMCs and being sufficient to modulate nonasthmatic cells to an asthmatic phenotype. MiR-155 is not predicted to directly target the 3′-untranslated region of COX-2 (43), but the miRNA might indirectly promote cytokine-responsive gene expression by targeting suppressor of cytokine signaling-1 and SH2-containing inositol-phosphatase-1 (SHIP1) (30, 44–46). Previously, miR-155–mediated repression of SHIP1 was observed to be responsible for miR-155–mediated stabilization of CXCL8 mRNA in cystic fibrosis–derived bronchial epithelial cells (IB3–1) (30). Posttranscriptional regulation of cytokine responsive gene expression (e.g., CXCL8 and COX-2) is highly conserved; thus, it is plausible that such a mechanism may be responsible for miR-155–mediated enhancement of COX-2 expression in hASMCs. MiR-155–mediated repression of SHIP1 led to enhanced activation of the phosphoinositide-3-kinase (PI3K)/AKT pathway in IB3–1 cells. In hASMCs, the PI3K/AKT pathway was observed to partly mediate cigarette smoke extract induction of COX-2 in hASMCs (47). Furthermore, AKT was previously observed to enhance the stability of COX-2 mRNA in rat intestinal epithelial cells (48). AKT regulates mRNA stability by phosphorylating and inhibiting the destabilizing function of two RNA binding proteins, butyrate response factor 1 and K homology splicing regulatory protein (49, 50). Repression of suppressor of cytokine signaling-1 by miR-155 may also enhance activation of the PI3K/AKT pathway. Thus, these studies indicate that miR-155 may enhance COX-2 expression in hASMCs by promoting PI3K/AKT inactivation of butyrate response factor 1 and K homology splicing regulatory protein, thereby enhancing COX-2 mRNA stability. Although our results point to a novel mechanism of regulation of COX-2 protein abundance by miR-155 in vitro, our results raise the question of whether COX-2 expression is enhanced in the airway smooth muscle of patients with asthma in vivo. It is known that there is elevated COX-2 immunoreactivity in the epithelium of patients with asthma, but no investigations have focused on the airway smooth muscle (14). COX-2–derived PGE₂ has been linked to β₂-adrenoceptor desensitization in hASMCs in vitro in response to cytokine treatment and in a coculture model of rhinovirus infection (1, 3–5). Furthermore, β₂-adrenoceptor agonist hyporesponsiveness has been observed clinically in patients with asthma and in postmortem tissue from patients with fatal asthma (2, 51). Altogether, the evidence points to autocrine PGE₂ signaling being enhanced in patients with asthma due to elevated expression of COX-2 and greater secretion of PGE₂ by hASMCs. Autocrine PGE₂ signaling may be further enhanced in patients with asthma due to increased E-prostanoid receptor surface expression based on previous observations by Burgess and colleagues (13).

In this study we report novel findings that cytokine stimulation of asthmatic hASMCs causes enhanced expression of COX-2 mRNA and protein, enhanced secretion of PGE₂, and enhanced expression of miR-155. We suggest that cytokine-mediated induction of miR-155 is sufficient to enhance COX-2 protein abundance in nonasthmatic hASMCs and propose that it may be responsible for the elevated expression of COX-2 that we observed in the asthmatic hASMCs. These results were previously presented at a scientific conference and as part of a dissertation (52, 53).