Lamyel, Fathi: Characterization of β-Adrenergic Mechanisms and their Interaction with Corticosteroids in Human Pulmonary Fibroblasts. - Bonn, 2014. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
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author = {{Fathi Lamyel}},
title = {Characterization of β-Adrenergic Mechanisms and their Interaction with Corticosteroids in Human Pulmonary Fibroblasts},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2014,
month = aug,

note = {Background: Both asthma and COPD are chronic lung diseases characterized by airway remodelling in which fibrotic alterations represents major part. This study focuses on the role of classical anti-obstructive drugs in the modulation of long-term remodelling processes in asthma and COPD, in an attempt to explore β-adrenergic mechanisms in lung fibroblasts.
Methods: Cell proliferation and collagen synthesis were analyzed by measuring incorporation of [3H]-thymidine and [3H]-proline, respectively. Expression of β-adrenoceptors and α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation was measured at the level of mRNA by semi-quantitative and real time RT-PCR and at the protein level by western blot analysis.
Results: In human lung fibroblasts (primary cells as well as MRC-5 cell line) stable expression of mRNA encoding β2-adrenoceptors, but no transcripts for β1- and β3-adrenoceptors could be demonstrated. β-Adrenoceptors agonists inhibited cell proliferation and collagen synthesis both in MRC-5 cells and PhLFb maximally by about 30-40 % and a detailed pharmacological characterization demonstrated that both effects were mediated via β2-adrenoceptors.
MRC-5 cells showed basal expression of α-SMA which was inhibited both at mRNA and protein level by about 50% by the β2-adrenoceptors selective agonist formoterol.
Both, PKA and Epac selective agonist mimicked the effect of β-adrenoceptors agonists on α-SMA mRNA and protein expression with some tendency for additive effects, whereas previous studies had shown that PKA and Epac agonists inhibited selectively either collagen synthesis or proliferation, respectively.
The effects of β-adrenoceptors agonists on proliferation, collagen synthesis and α-SMA expression were lost after exposure to transforming growth factor-β1 (TGF-β) which alone had no effect on proliferation, but caused a marked increase in collagen synthesis and a marked up-regulation of α-SMA expression.
After inhibition of mRNA synthesis by actinomycin D (30 μM),β2-adrenoceptors mRNA decreased with a half-life of 25 min, whereas inhibition of protein synthesis by cycloheximide (CHX), (30 μM) resulted in a rapid increase of β2-adrenoceptors mRNA (about 3- and 6- fold within 1.5 and 6.5 hrs, respectively). Indicating that β2-adrenoceptors expression is highly regulated at mRNA level.
TGF-β induced a marked and long lasting down-regulation of β2-adrenoceptors mRNA (about 90 % within 6 hrs). Also corticosteroids (dexamethasone and budesonide) alone caused a marked and long lasting up-regulation of β2-adrenoceptors mRNA, but they were not able to prevent or oppose the TGF-β induced down-regulation. Exposure to β-adrenoceptors agonists resulted in a rapid (maximum effect within 1 h), but transient up-regulation of β2-adrenoceptors mRNA level. This effect was mimicked by forskolin, a direct activator of adenylyl cyclase. Furthermore, as β-adrenoceptors agonists-induced up-regulation of β2-adrenoceptors mRNA also in presence of CHX, but not in presence of actinomycin D, it can be concluded that a cAMP direct stimulation of the expression of the β2-adrenoceptors gene may be involved.
In conclusion: Human lung fibroblasts exclusively express β2-adrenoceptors which mediate inhibition of various aspects of pro-fibrotic processes. The β2-adrenoceptors are highly regulated at mRNA level, and the up-regulation after β-adrenoceptors agonists-induced up-regulation of β2-adrenoceptors gene may be a mechanism to oppose agonist-induced receptor desensitization which may contribute to the maintenance of β2-adrenoceptors function during long term agonist exposure. Under clinical conditions, anti-fibrotic effects may associate the therapeutic effect of β2-adrenoceptors agonists treatment of asthma and COPD.},

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