Impaired E Prostanoid2 Expression and Resistance to Prostaglandin E2 in Nasal Polyp Fibroblasts from Subjects with Aspirin-Exacerbated Respiratory Disease

Abstract

Recurrent, rapidly growing nasal polyps are hallmarks of aspirin-exacerbated respiratory disease (AERD), although the mechanisms of polyp growth have not been identified. Fibroblasts are intimately involved in tissue remodeling, and the growth of fibroblasts is suppressed by prostaglandin E2 (PGE2), which elicits antiproliferative effects mediated through the E prostanoid (EP)2 receptor. We now report that cultured fibroblasts from the nasal polyps of subjects with AERD resist this antiproliferative effect. Fibroblasts from polyps of subjects with AERD resisted the antiproliferative actions of PGE2 and a selective EP2 agonist (P < 0.0001 at 1 μM) compared with nasal fibroblasts from aspirin-tolerant control subjects undergoing polypectomy or from healthy control subjects undergoing concha bullosa resections. Cell surface expression of the EP2 receptor protein was lower in fibroblasts from subjects with AERD than in fibroblasts from healthy control subjects and aspirin-tolerant subjects (P < 0.01 for both). Treatment of the fibroblasts with trichostatin A, a histone deacetylase inhibitor, significantly increased EP2 receptor mRNA in fibroblasts from AERD and aspirin-tolerant subjects but had no effect on cyclooxygenase-2, EP4, and microsomal PGE synthase 1 (mPGES-1) mRNA levels. Histone acetylation (H3K27ac) at the EP2 promoter correlated strongly with baseline EP2 mRNA (r = 0.80; P < 0.01). These studies suggest that the EP2 promotor is under epigenetic control, and one explanation for PGE2 resistance in AERD is an epigenetically mediated reduction of EP2 receptor expression, which could contribute to the refractory nasal polyposis typically observed in this syndrome.

Keywords: DNA methylation; E prostanoid type 2 receptor; aspirin-exacerbated respiratory disease; histone acetylation; prostaglandin E2.

Figure 1.

Fibroblast proliferation. Fibroblasts from nasal polyps of subjects with aspirin-exacerbated respiratory disease (AERD) (n = 11–12), aspirin-tolerant (AT) sinus disease (n = 6), or healthy control subjects (n = 5) were plated overnight and then serum-starved and treated with no stimulant, forskolin 100 μM, prostaglandin E₂ (PGE₂) (10 μM), or an E prostanoid 2 (EP₂) agonist (ONO-AE1–259, 0.01–1 μM) along with tritiated thymidine (³H-TdR) for 24 hours, and the incorporated ³H-TdR was recorded in counts per minute (cpm). Intrinsic baseline rate of proliferation (A) and percent inhibition of proliferation in response to forskolin, PGE₂, and the selective EP₂ agonist (B) are shown. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 2.

EP₂ receptor cell surface and mRNA expression on fibroblasts from healthy and AT control subjects compared with those from subjects with AERD. Cultured fibroblasts were serum starved for 48 hours and stained with EP₂ monoclonal antibodies. (A) A representative histogram of EP₂ cell surface expression of fibroblasts from a healthy control subject, an AT subject, and a patient with AERD are shown, along with the appropriate isotype control. (B) The percent of fibroblasts that expressed surface EP₂ (percent of EP₂-positive fibroblasts minus percent of isotype control positive fibroblasts) is shown from healthy control subjects (n = 5), AT control subjects (n = 3), and subjects with AERD (n = 7). (C) Mean fluorescence intensity (MFI) of EP₂ antibody minus MFI of isotype control of fibroblasts from healthy control subjects (n = 5), AT control subjects (n = 3), and subjects with AERD (n = 7). (D) Baseline EP₂ mRNA expression after serum starvation for 24 hours in healthy fibroblasts (n = 5), AT fibroblasts (n = 3), and AERD fibroblasts (n = 11). Data are expressed as individual subjects with group mean ± SEM. *P < 0.05 and **P < 0.01. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 3.

(A) To study the effects of IL-1β on mRNA expression of EP₂, EP₄, cyclooxygenase (COX)-2, and microsomal PGE synthase 1 (mPGES-1), cultured fibroblasts were serum starved and incubated in the presence or absence of IL-1β (1 ng/ml) for 24 hours (healthy, n = 4; AT, n = 3; AERD n = 11–13). Ratio paired t test was used to assess change from baseline. Data are expressed as fold change from baseline for individual subjects with group mean. (B) To study the effects of trichostatin A (TSA) on PGE₂ pathway gene expression, cultured fibroblasts from healthy control subjects (n = 5), AT subjects (n = 3), and subjects with AERD (n = 9–13) were serum starved and incubated in the presence of TSA or left untreated for 24 hours. EP₂, COX-2, mPGES-1, and EP₄ mRNA levels were determined by quantitative PCR with GAPDH used as a control and compared with untreated fibroblasts. Ratio paired t test was used to assess change from baseline. Data are expressed as fold change over baseline for individual subjects with group mean. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4.

Histone acetylation at PTGER2 in cultured nasal fibroblasts from AT control subjects and subjects with AERD. (A) Histone acetylation levels at PTGER2 promoter are shown for nine subjects. (B) Correlation between baseline EP₂ mRNA expression and baseline H3K27ac at PTGER2 in fibroblasts from all samples (healthy, n = 3; AERD, n = 6) was determined by Spearman’s correlation coefficient (r = 0.80; P < 0.01).