Abstract
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 β2-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 μM), or prostaglandin E2(1 μM) was accompanied by a reduction in cyclin D1 protein levels. The ERK kinase inhibitor PD98059 (3–30 μM) 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 μM). In contrast, neither albuterol (100 nM) nor PD98059 (30 μM) 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 inhibitorN-acetyl-Leu-Leu-leucinal (10 μM) attenuated the reduction in thrombin-stimulated cyclin D1 levels in ASM exposed to albuterol (100 nM), 8-bromo-cAMP (300 μM), or the phosphodiesterase inhibitor isobutylmethylxanthine (100 μM). Thus, the cAMP-induced arrest of ASM in the G1 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.
Footnotes
- Received April 19, 1999.
- Accepted July 30, 1999.
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Send reprint requests to: Alastair G. Stewart, Ph.D., Department of Pharmacology, University of Melbourne, Parkville, Victoria 3052, Australia. E-mail:a.stewart{at}pharmacology.unimelb.edu.au
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This work was supported in part by National Health Medical Research Council (Australia) and GlaxoWellcome (Uxbridge, UK).
- The American Society for Pharmacology and Experimental Therapeutics
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