Characterization of Expression and Functional Role of Endothelin-1 and its Receptors in Human Lung Fibroblasts

Abstract

<strong>Background</strong>: Background: Airway remodeling and fibrotic alterations are pathological key events determining the long-term outcome of chronic inflammatory and obstructive airway diseases such as lung fibrosis, bronchial asthma or COPD. In fact, these pathological conditions have a common tendency toward enhanced cellular activities mainly increased growth of connective tissue cells and excessive synthesis and secretion of matrix proteins. Lung fibroblasts are the most abundant cell type in connective tissue that produce and maintain extracellular matrix (ECM).<br /> Since endothelins (ETs), of which three isoforms (ET-1, ET-2 and ET-3) exist, can exert a number of effects which could promote remodeling processes, the present study aimed to characterize the endothelinergic system in human lung fibroblasts using the MRC-5 human lung fibroblast cell line and primary human lung fibroblasts in culture.<br /><strong>Methods</strong>: The expression of ETs and their receptors was analyzed at the mRNA level by quantitative real time PCR (qPCR) and at the peptide level by immune blot for the ET-A and ET-B receptor and by ELISA for ET-1. Functional effects of ET-1 on cell proliferation were quantified using a [³H]-thymidine incorporation assay and cell counts, those on collagen synthesis using a [³H]-proline incorporation assay, the QuickZyme assay and qPCR for collagen-1 α1 mRNA. In addition ET-1-induced changes on phenotype were detected by measuring α-smooth muscle actin (α-SMA) at the protein level.<br /><strong>Results</strong>: Human lung fibroblasts clearly express mRNA encoding ppET-1 and ppET-2 with the most dominantly expressed isoform being the ppET-1, but no transcript for ppET-3 could be demonstrated. Both subtypes of ET receptors, ET-A and ET-B were also expressed in human lung fibroblasts.<br /> The expression of ppET-1 mRNA and the release of ET-1 in human lung fibroblasts were significantly up-regulated by TGF-β. TGF-β induced an increase in ppET-1 gene transcription through its canonical Smad-pathway and was augmented by concomitant ET-A receptor activation. Moreover, the stimulatory effect of TGF-β on the expression of ET isoforms was confined to ET-1.<br /> After inhibition of RNA synthesis by actinomycin D, ppET-1 mRNA decreased with a half life of about 30 min, whereas inhibition of protein synthesis by cycloheximide resulted in a rapid, marked increase in ppET-1 mRNA. Indicating that the expression of ppET-1 is highly regulated at the transcription level. TGF-β induced up-regulation of ppET-1mRNA was prevented by actinomycin D, indicating that it is caused by increased gene transcription. On the other hand, in presence cycloheximide, the stimulatory effect of TGF-β was markedly augmented, suggest a direct regulation of ppET-1 mRNA gene expression by TGF-β signalling pathways and does not involve the up-regulation of other regulatory proteins. It is concluded that the expression of ppET-1 is under strong inhibitory control of not yet identified short living inhibitory proteins which also counteract TGF-β-induced up-regulation. ET-1 expression is regulated by stimulatory muscarinic receptors and inhibitory β2-adrenoceptors. The stimulatory effect of muscarinic receptors was opposed by concomitant activation of β2-adrenoceptors and by blocking of ET receptors. The inhibitory effect of β2-adrenoceptors on ppET-1 expression appears to be mediated via PKA, as it was mimicked by direct activation of PKA. The stimulatory effect of sub-maximally effective concentration of TGF-β on ppET-1 expression was opposed by β2-adrenoceptor-PKA pathway. However, excessive exposure to TGF-β results in loss of β-adrenoceptor expression and loss of function of its down-stream signaling.<br /> ET-1 stimulated proliferation and collagen synthesis in human lung fibroblasts and a detailed pharmacological characterization demonstrated that both effects were mediated via ET-A receptors. Activation of ERK-MAPK pathway is crucially involved in the stimulatory effect of ET-1 on collagen synthesis. ET-1 up-regulated the COL I-α1 mRNA, a major collagen type in lung fibroblasts, indicates that enhanced collagen gene transcription may at least contribute to the up-regulation of collagen synthesis. Blockade of ET receptors attenuated the muscarinic receptor-mediated and TGF-β-induced stimulation of collagen synthesis, indicating that the observed up-regulation of ET-1 is of functional significance and may contribute to the pro-fibrotic effect of cholinergic stimuli as well as to that of TGF-β. ET-1 induced differentiation of human lung fibroblasts into myofibroblasts, but was not involved in TGF-β-mediated human lung fibroblasts differentiation into myofibroblasts.<br /><strong>Conclusion</strong>: Human lung fibroblasts express a functional autocrine/paracrine endothelinergic system that appears to be involved in the regulation of pro-fibrotic features. ET-1 is the predominant ET isoform in human lung fibroblasts, which may in interaction with other mediators such as TGF-β as well as the adrenergic and cholinergic system, regulate pro-fibrotic features. Thus, ET-1 is highly up-regulated by TGF-β and via muscarinic cholinergic receptors and down-regulated via β2-adrenoceptors. ET-1 can mediate through ET-A receptors stimulatory effects on proliferation and collagen synthesis. In fact, there is no functional significance of ET-B receptors obtained although the expression of ET-B receptor was demonstrated, both at mRNA and protein levels.