Signal transduction pathways (MAPKs, NF-κB, and C/EBP) regulating COX-2 expression in nasal fibroblasts from asthma patients with aspirin intolerance

Abstract

Background: Recent studies have revealed that cyclooxygenase-2 (COX-2) expression is down-regulated in aspirin-induced asthma (AIA). Various signal pathways (MAPKs, NF-κB and C/EBP) are involved in COX-2 regulation.

Objective: To investigate the regulation of COX-2 expression through MAP-kinase pathway activation and nuclear factor translocation in aspirin-induced asthma (AIA).

Methods: Fibroblasts were isolated from specimens of nasal mucosa (NM, N = 5) and nasal polyps (NP, N = 5). After IL-1β (1 ng/ml) incubation, COX-2 and phosphorylated forms of ERK, JNK and p38 MAPK were measured by Western blot. MAPK's role in IL-1β-induced COX-2 expression was assessed by treating cells with ERK (PD98059), JNK (SP600125) and p38 MAPK (SB203580) inhibitors (0.1-10 µM) prior to IL-1β exposure. NF-κB and C/EBP nuclear translocation was measured by Western blot and TransAM® after IL-1β (10 ng/ml) exposure.

Results: No differences were observed in the MAPK phosphorylation time-course between NM and NP-AIA fibroblasts. The p38 MAPK inhibitor at 10 µM significantly reduced IL-1β-induced COX-2 expression in NM fibroblasts (85%). In NP-AIA fibroblasts the COX-2 inhibition (65%) at 1 and 10 µM was not statistically significant compared to non-treated cells. ERK and JNK inhibitors had no significant effect in either the NM or NP-AIA cultures. The effect of IL-1β on NF-κB and C/EBP subunits' nuclear translocation was similar between NM and NP-AIA fibroblasts.

Conclusions: These results suggest that p38 MAPK is the only MAPK involved in IL-1β-induced COX-2 expression. NM and NP-AIA fibroblasts have similar MAPK phosphorylation dynamics and nuclear factor translocation (NF-κB and C/EBP). COX-2 downregulation observed in AIA patients appears not to be caused by differences in MAPK dynamics or transcription factor translocation.

Figure 1. Time-course and dose-response of IL-1β in COX-1 and COX-2 protein expression.

Fibroblasts from nasal mucosa (NM) and nasal polyps from AIA patients (NP-AIA) were incubated at different times and concentrations with IL-1β. COX-1 expression was not altered by IL-1β treatment in either NM or NP-AIA fibroblasts. In NM fibroblasts, COX-2 expression was induced in a time-dependent and dose-response manner. In NP-AIA, COX-2 protein expression was not significantly increased, compared to baseline level. The image that is shown is representative of NM and NP-AIA Western blot.

Figure 2. Time-course of MAPK activation by IL-1β in nasal fibroblasts cultures.

Fibroblasts from nasal mucosa (NM, N = 4, black spots) and nasal polyps from AIA patients (NP-AIA, N = 4, white spots) were incubated with IL-1β (1 ng/ml) for 5 to 60 min. Phosphorylated and non-phosphorylated forms of p38 MAPK (A), JNK (B), and ERK (C) were measured by Western blot. Results are expressed as the ratio of phosphorylated versus non-phosphorylated MAPK forms. Graph shows individual experimental results and lines indicate the medians values. MAPK dynamic activation was not different (NS, Mann-Whitney U-test) between NM and NP-AIA fibroblasts. Insets show representative Western blot images of p38 MAPK, JNK, and ERK phosphorylation dynamics and β-actin as loading control.

Figure 3. Effect of MAPK specific inhibitors on COX-2 protein expression.

Fibroblasts from nasal mucosa (NM, N = 5, black spots) and nasal polyps from AIA patients (NP-AIA, N = 5, white spots) were pre-treated with p38 (A) MAPK (SB203580), (B) JNK (SP600125), and (C) ERK (PD98059) specific inhibitors at different concentrations (0.1–10 µM) for 1 h before addition of IL-1β (1 ng/ml) for 24 h. COX-2 and β-actin protein expression was analyzed by Western blot. Only the p38 MAPK inhibitor significantly blocked IL-1β-induced COX-2 expression in NM fibroblasts. Results are presented as COX-2/β-actin ratio. Graph shows individual experimental results and lines indicate the medians values. * p<0.05, † p = 0.06 compared to IL-1β treatment by Wilcoxon test.

Figure 4. Time-course of p65 and p50 nuclear translocation induced by IL-1β in nasal fibroblast cultures.

Fibroblasts from nasal mucosa (NM, N = 3, black spots) and nasal polyps from AIA patients (NP-AIA, N = 3, white spots) were incubated with IL-1β (10 ng/ml) for 5 to 60 min. p65 (A) and p50 (B) nuclear translocation were measured by TransAM® and Western blot. Graphs show the fold change increase from individual experimental results and lines indicate the medians values. Insets show representative Western blot images of p65 (A) and p50 (B) subunits from both NM and NP-AIA fibroblasts and the loading control β-actin. No significant differences (NS by Mann-Whitney U-test) were found at any time between NM and NP-AIA fibroblasts.