Role of mast cells and protease-activated receptor-2 in cyclooxygenase-2 expression in urothelial cells

Abstract

Mast cells have been shown to play a role in development and persistence of various inflammatory bladder disorders. Mast cell-derived tryptase specifically activates protease-activated receptor-2 (PAR-2), and PAR-2 is known to be involved in inflammation. We investigated whether mast cells participate in increase of cyclooxygenase-2 (COX-2) protein abundance in urothelium/suburothelium of bladders of mice subsequent to cyclophosphamide (CYP)-induced bladder inflammation. We also used primary cultures of human urothelial cells to investigate cellular mechanisms underlying activation of PAR-2 resulting in increased COX-2 expression. We found that treatment of mice with CYP (150 mg/kg ip) increased COX-2 protein abundance in bladder urothelium/suburothelium 3, 6, and 24 h after CYP (P < 0.01), and increased COX-2 protein abundance was prevented by treatment of mice with the mast cell stabilizer sodium cromolyn (10 mg/kg ip) for 4 consecutive days before CYP treatment. Incubation of freshly isolated mouse urothelium/suburothelium with a selective PAR-2 agonist, 2-furoyl-LIGRLO-amide (3 microM), also increased COX-2 protein abundance (P < 0.05). We further demonstrated that 2-furoyl-LIGRLO-amide (3 microM) increased COX-2 mRNA expression and protein abundance in primary cultures of human urothelial cells (P < 0.01), and the effects of PAR-2 activation were mediated primarily by the ERK1/2 MAP kinase pathway. These data indicate that there are functional interactions among mast cells, PAR-2 activation, and increased expression of COX-2 in bladder inflammation.

Fig. 1.

A: histological appearance (hematoxylin and eosin staining) of urothelium/suburothelium reveals that the contamination of detrusor was very limited. Scale bar, 50 μm. B: strong staining with a specific uroplakin antibody is shown in superficial urothelial cells. C: treatment of mice with cyclophosphamide (CYP; 150 mg/kg ip) for 3, 6, and 24 h induced an increase in cyclooxygenase-2 (COX-2) protein abundance in the bladder urothelium/suburothelium, respectively. D: similarly, treatment of mice with CYP induced an increase (6 and 24 h) in protease-activated receptor-2 (PAR-2) protein abundance in the bladder urothelium/suburothelium. Control mice received saline (ip). Relative COX-2 or PAR-2 protein abundance was analyzed by immunoblotting. Values determined by densitometry were normalized to those of GAPDH (loading control) in each sample. Data are means ± SE; n = 4–5. **P < 0.01 vs. control.

Fig. 2.

Treatment of mice with the mast cell stabilizer sodium cromolyn (CRO; 10 mg/kg) for 4 consecutive days inhibited CYP-induced increase in COX-2 protein abundance in bladder urothelium/suburothelium. Relative COX-2 protein abundance was analyzed 3 (A) and 6 h (B) after treatment with CYP. Values determined by densitometry were normalized to those of GAPDH (loading control) in each sample. Data are means ± SE; n = 4. **P < 0.01 vs. control. #P < 0.05; ##P < 0.01 vs. CYP-treated group.

Fig. 3.

A: representative images of cultured bladder urothelial cells stained with a specific monoclonal antibody against AE1/AE3 keratin. Scale bar, 50 μm. B: a normal mouse IgG was used instead of the specific antibody for immunocytochemistry as negative control. C: relative abundance of PAR-2 protein in urothelial cells was analyzed by immunoblotting. Treatment with a selective PAR-2 agonist for 6 h did not alter PAR-2 protein abundance (P > 0.05, n = 4). Values determined by densitometry were normalized to those of GAPDH (loading control) in each sample.

Fig. 4.

A: treatment of primary cultures of human urothelial cells with a selective PAR-2 agonist (3 μM) altered expression of COX-2 mRNA with significant increase at 30 min and at 1 and 2 h. Data are means ± SE; n = 6. **P < 0.01 vs. control. B: PAR-2 agonist (3 μM) induced phosphorylation of ERK1/2, which was abolished by U0126 (10 μM), a selective ERK1/2 inhibitor. A representative example of the results of 3 independently performed experiments is shown. C: treatment with U0126 (10 μM) prevented PAR-2 agonist-induced increase in expression of COX-2 mRNA at 1 h. Data are means ± SE; n = 6. **P < 0.01 vs. control. ##P < 0.01 vs. PAR-2 agonist-treated group. D: treatment of PAR-2 agonist (3 μM) for 6 h also increased COX-2 protein abundance, and this effect of PAR-2 was prevented by U0126 (10 μM). Data are means ± SE; n = 4. **P < 0.01 vs. control. #P < 0.05 vs. PAR-2 agonist-treated group.