Characterization of β-Adrenergic Mechanisms and their Interaction with Corticosteroids in Human Pulmonary Fibroblasts

Abstract

<strong>Background</strong>: 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. <br /><strong>Methods</strong>: 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. <br /><strong>Results</strong>: 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. <br /> 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. <br /> 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. <br /> 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. <br /> 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. <br /> 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. <br /> 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.