We have previously shown that glucocorticoids inhibit mitogen-stimulated proliferation of human cultured airway smooth muscle (ASM) cells. The present study analyzed the effect of glucocorticoids on key regulatory pathways leading to passage of cells through the restriction point of the cell cycle, including those mediated by extracellular-regulated kinases (ERK) 1 and 2; the ERK upstream regulator MAPK kinase (MEK1); cyclin D1 levels; and levels and phosphorylation of retinoblastoma protein (pRb). Fluticasone propionate, a new inhaled glucocorticoid, was at least 10-fold more potent than dexamethasone in inhibiting thrombin-stimulated DNA synthesis and increases in cell number. Thrombin-stimulated increases in the levels and hyperphosphorylation of pRb were inhibited by glucocorticoids, which also reduced thrombin-stimulated cyclin D1 protein and messenger RNA (mRNA) levels. PD98059 (10 microM), an inhibitor of MEK1 activation, markedly attenuated thrombin stimulation of ERK activity and phosphorylation, DNA synthesis, and cyclin D1 levels. However, glucocorticoids had no effect on ERK activity or phosphorylation at 5 min, 2 h, or 12 h after addition of thrombin. In conclusion, glucocorticoid-induced reduction of cyclin D1 mRNA and protein levels, and of pRb phosphorylation, is sufficient to account for inhibition of ASM proliferation. Furthermore, these inhibitory effects of glucocorticoids on cyclin D1 and pRb occur on a component of the mitogen signaling cascade that is either downstream of or parallel to the ERK pathway.

1 The relationship between persistent ERK (extracellular signal-regulated kinase) activity, cyclin D1 protein and mRNA levels and cell cycle progression in human cultured airway smooth muscle was examined in response to stimulation by ET-1 (endothelin-1), thrombin and bFGF (basic ®broblast growth factor). 2 Thrombin (0.3 and 3 u ml 71 ) and bFGF (0.3 and 3 nM) increased ERK activity for more than 2 h and increased cell number, whereas ET-1 (100 nM) transiently stimulated ERK activity and was non-mitogenic. 3 The MEK1 (mitogen-activated ERK kinase) inhibitor, PD 98059 (30 mM), inhibited both ERK phosphorylation and activity, and either prevented (thrombin 0.3 and 3 u ml 71, bFGF 300 pM) or attenuated (bFGF 3 nM) DNA synthesis. 4 Thrombin and bFGF increased both cyclin D1 mRNA and protein levels. PD 98059 decreased cyclin D1 protein levels stimulated by the lower but not higher thrombin concentrations. Moreover, increases in cyclin D1 mRNA levels were unaected by PD 98059 pretreatment, irrespective of the mitogen or its concentration, suggesting that inhibition of cyclin D1 protein levels occurred by a post-transcriptional mechanism. 5 These ®ndings indicate that the control of cyclin D1 protein levels may occur independently of the MEK1/ERK signalling pathways. The inhibition of S phase entry by PD 98059 at higher thrombin concentrations appears to result from eects on pathways downstream or parallel to those regulating cyclin D1 protein levels. These ®ndings suggest heterogeneity in the signalling of DNA synthesis in human cultured airway smooth muscle.

Hyperplasia of airway smooth muscle (ASM) contributes to the airway hyperresponsiveness that characterizes asthma. We have investigated the relationship between cAMP-induced growth arrest of ASM cells and thrombin-stimulated, extracellular-regulated protein kinase (ERK) activity, cyclin D1, and the restriction protein retinoblastoma. The beta(2)-adrenergic receptor agonist albuterol (100 nM) inhibited DNA synthesis after incubation with ASM for periods as brief as 1 h when these coincided with the timing of the restriction point. Inhibition of thrombin-stimulated DNA synthesis by albuterol (1-100 nM), 8-bromo-cAMP (300 microM), or prostaglandin E(2) (1 microM) was accompanied by a reduction in cyclin D1 protein levels. The ERK kinase inhibitor PD98059 (3-30 microM) attenuated thrombin-stimulated ERK phosphorylation and activity and the increase in cyclin D1 protein levels, as did albuterol (1-100 nM) or 8-bromo-cAMP (300 microM). In contrast, neither albuterol (100 nM) nor PD98059 (30 microM) reduced cyclin D1 mRNA levels between 4 and 20 h after thrombin addition, which suggests that elevation of cAMP regulates cyclin D1 by a post transcriptional mechanism. The proteasome inhibitor MG132 (30 and 100 nM) and the calpain I inhibitor N-acetyl-Leu-Leu-leucinal (10 microM) attenuated the reduction in thrombin-stimulated cyclin D1 levels in ASM exposed to albuterol (100 nM), 8-bromo-cAMP (300 microM), or the phosphodiesterase inhibitor isobutylmethylxanthine (100 microM). Thus, the cAMP-induced arrest of ASM in the G(1) phase of the cell cycle is associated with a proteasomal degradation of cyclin D1 protein and a reduced protein retinoblastoma phosphorylation that prevents passage through the restriction point.