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Keywords:

  • endothelin;
  • ET-1;
  • ET-A and ET-B receptor;
  • collagen synthesis;
  • lung fibroblasts;
  • airway remodelling;
  • myofibroblast;
  • ERK MAPK;
  • TGF-β;
  • pertussis toxin;
  • bosentan;
  • dynamic mass redistribution (DMR)

Background and Purpose

Since 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 Approach

MRC-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 [3H]-thymidine was determined as measure of proliferation and that of [3H]-proline for collagen synthesis. Phospho-p42/44 MAP kinase was determined by Western blot.

Key Results

ET-1 is the predominant ET in human lung fibroblasts (hLF), and TGF-β 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 Implications

hLFb 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.