Mechanisms of DiseaseInteraction between glucocorticoids and β2 agonists on bronchial airway smooth muscle cells through synchronised cellular signalling
Introduction
Glucocorticoids and long-acting β2 agonists are widely used in treatment of asthma, and results of studies lend support to the combination of both classes of drugs in the long-term treatment of the condition. The combination of glucocorticoids and long-acting β2 agonists results in better symptom control and reduced airway inflammation than raised doses of glucocorticoids alone, suggesting that the two classes of drugs interact at an as yet undefined molecular level.1, 2, 3, 4, 5
Glucocorticoids work by binding to the cytosolic glucocorticoid receptor, inducing the formation of a dimer that is translocated into the nucleus and acts as a transcription factor6, 7 β2 agonists bind to the G-protein coupled β2 adrenoceptor in the cell membrane, and induce a cAMP/protein kinase signalling cascade.8
An important aim in asthma therapy is to prevent the accelerated growth of bronchial smooth muscle cells, which leads to hyperplasia and bronchial hyper-reactivity.9, 10 Glucocorticoids and long-acting β2 agonists inhibit the proliferation of human airway smooth muscle cells in vitro. However, the cellular signalling pathway involved has not been described in detail.11, 12 In hepatoma cells derived from mice, glucocorticoids inhibit proliferation via activation of the ccaat-enhancer binding protein (cebp)-α and, subsequently, of P21(waf)/(cip)13. In lung mesenchymal cells from human beings, Rüdiger and colleagues14 showed that the antiproliferative effect of glucocorticoids depends on C/EBP-α forming a complex with the glucocorticoid receptor, resulting in induction of p21(Wafl/Cipl). Our aim was to investigate how the combination of glucocorticoids with β2 agonists modulates cell proliferation.
Section snippets
Methods
We established eight primary cell cultures of human bronchial smooth muscle cells from muscle bundles removed from bronchial explants obtained from lung resections (surgical cancer therapy or lung transplantation) as previously described.9 Treatment and experiments were done with 80% confluent cell cultures between passages five and eight.
24 h before stimulation of cells, we replaced growth medium with low serum medium (0·1% fetal bovine serum [FBS]). We added formoterol or budesonide, or both
Results
Figure 1 shows activation of the glucocorticoid receptor and C/EBP-α in human bronchial airway smooth muscle cells. The non-activated glucocorticoid receptor was located in the cytosol of the cell (figure 1A), whereas once activated by formoterol and budesonide the glucocorticoid receptor translocated into the nucleus (figure 1B). We measured the kinetics of glucocorticoid-receptor activation by each drug alone (10−10 mol/L and 10−8 mol/L) or in combination (both 10−10 mol/L) as the proportion
Discussion
Our findings show that glucocorticoids and β2 agonists activate the glucocorticoid receptor and C/EBP-α, thereby inhibiting the proliferation of human bronchial airway smooth muscle cells via the action of p21(Wafl/Cipl). Furthermore, when given alone, formoterol and budesonide activate the two transcription factors with different potency and kinetics. In combination, the drugs lead to a simultaneous activation of the glucocorticoid receptor and C/EBP-α, resulting in a synergistic stimulatory
GLOSSARY
- antisense oligonucleotide
- A short synthetic DNA sequence complementary to, and binding to, a specific messenger RNA, which upon binding inhibits translation into protein.
- ccaat-enhancer binding proteins (c/ebp)
- A family of transcription factors which are involved in many cell processes, including control of cell proliferation.
- electrophoretic mobility shift assay (emsa)
- A method used to characterise specific proteins that recognise and bind to a DNA sequence in a gene promoter, thereby regulating
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