Dual regulation of ß2-adrenoceptor messenger RNA expression in human lung fibroblasts by ß2-cAMP signaling; delayed upregulated inhibitors oppose a rapid in onset, direct stimulation of gene expression.

Based on their bronchodilatory effect, ß2-adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and COPD. As treatment with ß2-adrenoceptor agonists has been associated with worsening of airway hyper-reactivity, possibly because of loss of ß-adrenoceptor function, molecular mechanism of the regulation of ß2-adrenoceptor expression were studied. MRC-5 human lung fibroblasts were cultured in absence or presence of test substances followed by ß2-adrenoceptor messenger RNA (mRNA) determination by qPCR. After inhibition of mRNA synthesis by actinomycin D, ß2-adrenoceptor mRNA decreased with a half-life of 23 min, whereas inhibition of protein synthesis by cycloheximide caused an about 5- and 6-fold increase within 1.5 and 4 h, respectively. ß2-Adrenoceptor mRNA was increased by about 100 % after 1 h exposure to formoterol or olodaterol but decreased by about 60 % after 4 h agonist exposure. Both effects of ß2-adrenoceptor agonists were mimicked by forskolin, a direct activator of adenylyl cyclase and cholera toxin, which stimulates adenylyl cyclase by permanent activation of Gs. ß2-Adrenoceptor agonist-induced upregulation of ß2-adrenoceptor mRNA was blocked by the ß2-adrenoceptor antagonist ICI 118551 and prevented by actinomycin D, but not by cycloheximide. Moreover, in presence of cycloheximide, ß2-adrenoceptor agonist-induced reduction in ß2-adrenoceptor mRNA was converted into stimulation, resulting in a more than 10-fold increase. In conclusion, expression of ß2-adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. The ß2-adrenoceptor gene is under strong inhibitory control of short-living suppressor proteins. ß2-Adrenoceptor activation induces via adenylyl cyclase - cyclic adenosine monophosphate (cAMP) signaling a rapid in onset direct stimulation of the ß2-adrenoceptor gene transcription, an effect opposed by a delayed upregulation of inhibitory factors.

The ß2-subtype of adrenoceptors mediates inhibition of pro-fibrotic events in human lung fibroblasts.

Fibrosis is part of airway remodelling observed in bronchial asthma and COPD. Pro-fibrotic activity of lung fibroblasts may be suppressed by ß-adrenoceptor activation. We aimed, first, to characterise the expression pattern of ß-adrenoceptor subtypes in human lung fibroblasts and, second, to probe ß-adrenoceptor signalling with an emphasis on anti-fibrotic actions. Using reverse transcription PCR, messenger RNA (mRNA) encoding ß(2)-adrenoceptors was detected in MRC-5, HEL-299 and primary human lung fibroblasts, whereas transcripts for ß(1)- and ß(3)-adrenoceptors were not found. Real-time measurement of dynamic mass redistribution in MRC-5 cells revealed ß-agonist-induced G(s)-signalling. Proliferation of MRC-5 cells (determined by [(3)H]-thymidine incorporation) was significantly inhibited by ß-agonists including the ß(2)-selective agonist formoterol (-logIC(50), 10.2) and olodaterol (-logIC(50), 10.6). Formoterol's effect was insensitive to ß(1)-antagonism (GCP 20712, 3 µM), but sensitive to ß(2)-antagonism (ICI 118,551; apparent, pA (2), 9.6). Collagen synthesis in MRC-5 cells (determined by [(3)H]-proline incorporation) was inhibited by ß-agonists including formoterol (-logIC(50), 10.0) and olodaterol (-logIC(50), 10.3) in a ß(2)-blocker-sensitive manner. α-Smooth muscle actin, a marker of myo-fibroblast differentiation, was down-regulated at the mRNA and the protein level by about 50% following 24 and 48 h exposure to 1 nM formoterol, a maximally active concentration. In conclusion, human lung fibroblasts exclusively express ß(2)-adrenoceptors and these mediate inhibition of various markers of pro-fibrotic cellular activity. Under clinical conditions, anti-fibrotic actions may accompany the therapeutic effect of long-term ß(2)-agonist treatment of bronchial asthma and COPD.