Role of ERK MAP kinases in responses of cultured human airway smooth muscle cells to IL-1β

We have previously reported that interleukin (IL)-1β causes β- adrenergic hyporesponsiveness in cultured human airway smooth muscle cells by increasing cyclooxygenase-2 (COX-2) expression and prostanoid formation. The purpose of this study was to determine whether extracellular signal-regulated kinases (ERKs) are involved in these events. Levels of phosphorylated ERK (p42 and p44) increased 8.3- and 13-fold, respectively, 15 min after treatment with IL-1β (20 ng/ml) alone. Pretreating cells with the mitogen- activated protein kinase kinase inhibitor PD98059 or U-126 (2 h before IL-1β treatment) decreased ERK phosphorylation. IL-1β (20 ng/ml for 22 h) alone caused a marked induction of COX-2 and increased basal PGE₂ release 28-fold (P < 0.001). PD-98059 (100 μM) and U-126 (10 μM) each decreased COX-2 expression when administered before IL-1β treatment. In control cells, PD- 98059 and U-126 had no effect on basal or arachidonic acid (AA; 10 μM)- stimulated PGE2 release, but both inhibitors caused a significant decrease in bradykinin (BK; 1 μM)-stimulated PGE₂ release, consistent with a role for ERK in the activation of phospholipase A₂ by BK. In IL-1β-treated cells, prior administration of PD-98059 caused 81, 92 and 40% decreases in basal and BK and AA-stimulated PGE₂ release, respectively (P < 0.01), whereas administration of PD-98059 20 h after IL-1β resulted in only 38 and 43% decreases in basal and BK-stimulated PGE₂ release, respectively (P < 0.02) and had no effect on AA-stimulated PGE₂ release. IL-1β attenuated isoproterenol-induced decreases in human airway smooth muscle stiffness as measured by magnetic twisting cytometry, and PD-98059 or U-126 abolished this effect in a concentration-dependent manner. These results are consistent with the hypothesis that ERKs are involved early in the signal transduction pathway through which IL-1β induces PGE₂ synthesis and β-adrenergic hyporesponsiveness and that ERKs act by inducing COX-2 and activating phospholipase A₂.