Beta2-adrenergic receptor agonists and cAMP arrest human cultured airway smooth muscle cells in the G(1) phase of the cell cycle: role of proteasome degradation of cyclin D1.
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.
Stewart
A G
AG
Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia. a.stewart@pharmacology.unimelb.edu.au
Harris
T
T
Fernandes
D J
DJ
Schachte
L C
LC
Koutsoubos
V
V
Guida
E
E
Ravenhall
C E
CE
Vadiveloo
P
P
Wilson
J W
JW
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000420:Albuterol / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D015232:Dinoprostone / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D016193:G1 Phase / Q000187:drug effects*; D005786:Gene Expression Regulation; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism*; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D016196:S Phase / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors
D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists; D005419:Flavonoids; D007976:Leupeptins; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; D019938:Cyclin D1; D015124:8-Bromo Cyclic Adenosine Monophosphate; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D013917:Thrombin; D002154:Calpain; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D015232:Dinoprostone; D000420:Albuterol; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one