Ligand-independent Activation of the Glucocorticoid Receptor by β2-Adrenergic Receptor Agonists in Primary Human Lung Fibroblasts and Vascular Smooth Muscle Cells

Abstract

The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor present in most cell types. Upon ligand binding, the GR is activated and translocates into the nucleus where it transmits the anti-inflammatory actions of glucocorticoids. Here, we describe the ligand-independent activation of GR by the ␤ 2 -adrenergic receptor (␤ 2 -AR) agonists, salbutamol and salmeterol, in primary human lung fibroblasts and vascular smooth muscle cells. Immunohistochemistry demonstrated expression of GR and the ␤ 2 -AR by fibroblasts and vascular smooth muscle cells. Treatment of the cells with the ␤ 2 -AR agonists, salbutamol or salmeterol, resulted in translocation of GR into the nucleus beginning at 30 min, as shown by immunohistochemistry and Western blotting of cytosolic and nuclear cell extracts. In comparison, activation of GR induced by the corticosteroids dexamethasone and fluticasone occurred at the same time after treatment (30 min) but resulted in a more complete depletion of GR from the cytosolic compartment. Electrophoretic mobility shift assays confirmed that nuclear GR, activated by both ␤ 2 -AR agonists and glucocorticoids, actively bound to the GR consensus sequence (GR element). Functional activation of the GR was confirmed by a Luciferase reporter gene assay, using a GR driven promoter fragment from the p21 (WAF1/CIP1) gene. The effects of the ␤ 2 -AR agonists, salbutamol and salmeterol, were dependent upon binding to the ␤ 2 -AR, because blocking of ␤ 2 -AR with propranolol abrogated GR activation. GR activation appeared to involve cAMP. In summary, these data show that ␤ 2 -AR agonists are potent activators of GR. Ligandindependent activation of GR by ␤ 2 -AR agonists may substantially mediate the anti-inflammatory actions of these drugs observed in vitro and in vivo.

Synergistic effects of fluticasone propionate and salmeterol on in vitro T-cell activation and apoptosis in asthma

“…These findings suggest that the synergistic effects on PBT apoptosis and on the phosphorylation process of IkBa might occur at the level of the nuclear translocation of the GR. In contrast to Eickelberg et al, 11 salmeterol alone did not induce nuclear translocation of the GR. Importantly, it must be noted, this apparent discrepancy might be due to the lower b 2 receptor expression in PBTs compared with that seen in fibroblasts and vascular smooth muscle cells.…”

“…In a model of fibroblasts and vascular smooth muscle cells, LABAs upregulate the activity of ICSs by promoting the translocation of glucocorticoid receptor (GR) into the nuclei of these cells. 11 We have previously demonstrated that fluticasone propionate (FP) is able to induce peripheral blood T-cell apoptosis in asthma 7 and that steroids are able to reduce the NF-kB activation in PBTs. 12 Despite this, the effects of the combination of ICSs and LABAs on T cells has not yet been firmly established.…”

Fluticasone and salmeterol downregulatein vitro, fibroblast proliferation and ICAM-1 or H-CAM expression

“…These findings are in agreement with the results of a recent report, showing inhibition of ICAM-1 expression by human lung fibroblast primary cultures in the presence of budesonide and formoterol [31]. Indeed, b 2 -adrenergic receptor agonists may also activate glucocorticoid receptors in primary human lung fibroblasts [32].…”

How Do Corticosteroids Work in Asthma?

“…Corticosteroids increase the expression of ␤ 2 -adrenergic receptors in the lung and prevent their downregulation and uncoupling in response to ␤ 2 -agonists (42)(43)(44). Recent studies also show that ␤ 2 -agonists enhance the action of corticosteroids, with an increase in nuclear translocation of glucocorticoid receptors in vitro (45) and enhanced suppression of inflam- Endothelin-1 * GM-CSF ϭ granulocyte-macrophage colony-stimulating hormone; ICAM ϭ intercellular adhesion molecule-1; IL ϭ interleukin; MCP ϭ monocyte chemoattractant protein; MIP ϭ macrophage inflammatory protein; NF-B ϭ nuclear factor-B; RANTES ϭ regulated upon activation, normal cell expressed and secreted; SCF ϭ stem-cell factor; TNF-␣ ϭ tumor necrosis factor-␣; VCAM-1 ϭ vascular cell adhesion molecule-1.…”