Glucocorticoids Inhibit Proliferation, Cyclin D1 Expression, and Retinoblastoma Protein Phosphorylation, but Not Activity of the Extracellular-Regulated Kinases in Human Cultured Airway Smooth Muscle

Abstract

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.

Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma

“…However, no effect of salbutamol was found on cyclin D 1 mRNA, suggesting a post-transcriptional effect on cyclin D 1 , perhaps involving accelerated degradation of the cyclin D 1 protein [159]. In contrast to the lack of effect of glucocorticoids on ERK activity [155], salbutamol prevented ERK activation between 5 min and 8 h following stimulation with a-thrombin, consistent with earlier reports that sustained ERK activation throughout G 1 is necessary for cell cycle traversal [74,75,78]. An additional mechanism that was not investigated in this study, but has been reported previously by these authors [160], was the salbutamol-induced expression of p27 kip1 , a cdk protein inhibitor which prevents phosphorylation of pRb and cell cycle progression by inhibiting cdk activity in the mitogen-activated cyclin D 1 /cdk4 complex.…”

“…Consistent with the induction of growth-arrest in G 1 and attenuated phosphorylation of pRb, glucocorticoids also reduced levels of a-thrombin-stimulated increases in cyclin D 1 expression. In the same study, dexamethasone was without effect on the activation of ERK by a-thrombin, suggesting that the major effect of glucocorticoids in preventing S phase traversal in these cells was parallel to, or downstream of, ERK activation [155]. However, the events that link activation of ERK to cyclin D 1 expression in airway smooth muscle are unknown, although activated glucocorticoid receptors are known to form a complex with AP-1, perhaps preventing its action on genes required for cell cycle progression to the S phase.…”

“…In cell culture-based systems, however, glucocorticoids inhibit mitogen-stimulated proliferation of smooth muscle derived from bovine [152], rabbit [153] and human airways [154]. A recent study by STEWART and coworkers [155] has addressed the possible molecular mechanisms by which glucocorticoids could prevent induction of DNA synthesis by a-thrombin. Glucocorticoids were found to inhibit the passage of human airway smooth muscle cells through the restriction point (the stage in the cell cycle beyond which there is commitment of a cell to enter the S phase and to one round of DNA replication) by inducing a noncytotoxic, nonapoptotic arrest in the G 1 phase of the cell cycle [155].…”

“…A recent study by STEWART and coworkers [155] has addressed the possible molecular mechanisms by which glucocorticoids could prevent induction of DNA synthesis by a-thrombin. Glucocorticoids were found to inhibit the passage of human airway smooth muscle cells through the restriction point (the stage in the cell cycle beyond which there is commitment of a cell to enter the S phase and to one round of DNA replication) by inducing a noncytotoxic, nonapoptotic arrest in the G 1 phase of the cell cycle [155]. This was associated with reduced levels of a-thrombinstimulated phosphorylation of pRb by dexamethasone.…”

Airway smooth muscle cells: contributing to and regulating airway mucosal inflammation?

“…Corticosteroids can inhibit the induced release of RANTES, IL-8, GM-CSF and MCP-1 from airway smooth muscle cells in vitro, although they are not effective in inhibiting eotaxin release. In addition, they inhibit thrombin-induced airway smooth muscle proliferation through an inhibition of cyclin protein expression [84,85].…”