Fibrosis is part of airway remodelling observed in bronchial asthma and COPD. Pro-fibrotic activity of lung fibroblasts may be suppressed by β-adrenoceptor activation. We aimed, first, to characterise the expression pattern of β-adrenoceptor subtypes in human lung fibroblasts and, second, to probe β-adrenoceptor signalling with an emphasis on anti-fibrotic actions. Using reverse transcription PCR, messenger RNA (mRNA) encoding β 2 -adrenoceptors was detected in MRC-5, HEL-299 and primary human lung fibroblasts, whereas transcripts for β 1 -and β 3 -adrenoceptors were not found. Real-time measurement of dynamic mass redistribution in MRC-5 cells revealed β-agonistinduced G s -signalling. Proliferation of MRC-5 cells (determined by [ 3 H]-thymidine incorporation) was significantly inhibited by β-agonists including the β 2 -selective agonist formoterol (−logIC 50 , 10.2) and olodaterol (−logIC 50 , 10.6). Formoterol's effect was insensitive to β 1 -antagonism (GCP 20712, 3 μM), but sensitive to β 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 β-agonists including formoterol (−logIC 50 , 10.0) and olodaterol (−logIC 50 , 10.3) in a β 2 -blockersensitive manner. α-Smooth muscle actin, a marker of myofibroblast 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 β 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 β 2 -agonist treatment of bronchial asthma and COPD.
BACKGROUND AND PURPOSESince endothelin (ET) may act as pro-fibrotic mediator, expression and release of ET isoforms, their receptors and potential pro-fibrotic ET effects were studied in human lung fibroblasts. EXPERIMENTAL APPROACHMRC-5 and primary human lung fibroblasts (phLFb) were cultured. Expression of prepro-ET isoforms was determined by qPCR and release of ET-1 by ELISA. ET receptor function was analysed by real-time measurement of dynamic mass redistribution (DMR). Incorporation of [ KEY RESULTSET-1 is the predominant ET in human lung fibroblasts (hLF), and TGF-b caused a further, selective and sustained up-regulation of ET-1 resulting in increased extracellular ET-1 accumulation. hLFb express mRNA encoding ET-A and ET-B receptors. Expression of both receptors was confirmed at protein level. ET-1 induced marked DMR signals, an effect that involved ET-A and ET-B receptors. Stimulatory effects of ET-1 on hLFb proliferation and collagen synthesis were mediated exclusively via ET-A receptors. ET-1, again via ET-A receptors, induced rapid activation of ERK MAPK, shown to be a crucial cellular signal in ET-1-induced collagen synthesis. ET-1-induced activation of ERK and collagen synthesis was, in contrast to corresponding effect of a muscarinic agonist, largely insensitive to pertussis toxin. CONCLUSIONS AND IMPLICATIONShLFb are endowed with all elements necessary to build a functional autocrine/paracrine endothelinergic system, which appears to drive pro-fibrotic airway and lung remodelling processes, effects for which only ET-A, but not ET-B receptors appear to be of significance.
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