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Endothelins are a family of small peptides of 21 amino acids (ET-1, -2 and -3). They are synthesized as large precursor proteins (prepro-ET) that are processed by a cascade of different proteases resulting in big-ET and finally the active ET (for review, see, e.g. Polikepahad et al., 2006; Khimji and Rockey, 2010). The last step is mediated by endothelin-converting enzyme (ECE), of which several isoenzymes have been described, some of them located intracellularly and some appear to act as ectoenzymes on the extracellular cell surface (for review, see, e.g. Battistini and Jeng, 2001; Corder, 2001). ET-1 is one of the most potent constrictors of vascular smooth muscle, and its role in the pathophysiology of pulmonary hypertension has extensively been studied (for review, see Shao et al., 2011). In addition, ET-1 is a potent constrictor of airway smooth muscle and may exert various pro-fibrotic effects. Therefore, it is also considered to participate in the pathogenesis of pulmonary fibrosis and obstructive airway disease (e.g. Chalmers et al., 1997; Goldie and Henry, 1999; Polikepahad et al., 2006; Ross et al., 2010, Swigris and Brown, 2010). There is evidence that ET-1 is synthesized and released by different pulmonary cells, among them pulmonary fibroblasts and airway epithelial cells, and increased levels of ET-1 were observed in asthmatic patients (Pégorier et al., 2007). Moreover, the levels of ET-1 in exhaled breath condensate appear to correlate with the severity of asthma (Zietkowski et al., 2008). In different experimental models, ET-1 was shown to promote pulmonary inflammatory reactions, particularly cellular infiltration (e.g. Bhavsar et al., 2008a,b; Landgraf and Jancar, 2008) and the release of cytokines (e.g. Gallelli et al., 2005; Iwata et al., 2009) and prostanoids (Juergens et al., 2008). Furthermore, as ET-1 acts also as proliferative stimulus in airway smooth muscle (Panettieri et al., 1996) and fibroblasts (Gallelli et al., 2005), it may significantly contribute to airway and lung remodelling processes. Although there are several reports describing pro-fibrotics effects of ET-1 in rat and human pulmonary fibroblasts (e.g. Gallelli et al., 2005; Préfontaine et al., 2008), a detailed analysis of the expression of ET isoforms and ET receptors and their potential regulation in human pulmonary fibroblasts as well as a detailed pharmacological characterization of their functions are still missing.
Therefore, the present study aimed to explore systematically the expression and release of ET isoforms, their receptors, as well as potential functional effects of ET and ET-activated cellular signals in human lung fibroblasts. The present data show that human lung fibroblasts are endowed with all elements necessary to build a functional autocrine/paracrine endothelinergic system that appears to be involved in the regulation pro-fibrotic features.
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The present study shows that human lung fibroblasts are endowed with all elements of a functional autocrine/paracrine endothelinergic system. They express prepro-ET and are able to process it to the active mediator. They also express functional ET receptors that appear to regulate ‘pro-fibrotic’ features.
MRC-5 cells and phLFb express mRNA for prepro-ET-1 and prepro-ET-2, but no transcript for prepro-ET-3. However, in both MRC-5 cells and primary cells, mRNA levels for prepro-ET-1 largely exceeded those for prepro-ET-2, indicating that ET-1 is the predominant ET in human lung fibroblasts. Under standard culture conditions, mRNA levels for prepro-ET-1 and prepro-ET-2 were somewhat higher in MRC-5 cells than in phLFb. However, the expression of prepro-ET-1 mRNA is highly up-regulated by TGF-β, up to about ninefold in MRC-5 and more than 15-fold in phLFb, resulting in nearly similar expression levels in both cell types. The stimulatory effect of TGF-β was rapid in onset and did not show any fatigue during prolonged exposure suggesting that pathological conditions with prolonged elevation of TGF-β might be accompanied by a sustained up-regulation of ET-1 expression.
The stimulatory effect of TGF-β on ET expression is confined to ET-1, as TGF-β did not induce the expression of prepro-ET-3 and caused even a reduction of the already low levels of prepro-ET-2 (Figure 2B).
Expression of ET-1 mRNA in human lung fibroblasts appears to be highly regulated. As a consequence of the short half-life (30 min), prepro-ET-1 mRNA levels are expected to reflect immediately changes in prepro-ET-1 gene transcription. In fact, prepro-ET-1 mRNA levels were increased about sevenfold already after 1 h exposure to TGF-β (Figures 3 and 4).
TGF-β-induced up-regulation of prepro-ET-1 mRNA was prevented by actinomycin D, indicating that it is caused by increased gene transcription. The rapid onset of the TGF-β effect and the observation that it occurred also in presence of cycloheximide suggest a direct regulation of prepro-ET-1 mRNA gene expression by TGF-β signalling pathways. That cycloheximide caused a rapid increase in prepro-ET-1 mRNA indicates that prepro-ET-1 gene expression is under inhibitory control of short-living regulatory peptides, which appear also to oppose the stimulatory effect of TGF-β, as the TGF-β response was markedly enhanced in presence of cycloheximide (Figure 4).
Accumulation of the biological active ET-1 in culture media could also be detected and exposure to TGF-β resulted in increased levels. Strikingly, the maximum effect of TGF-β on ET-1 accumulation was seen at concentrations that induced only sub-maximal increase in prepro-ET-1 mRNA (compare Figures 2A and 6A). A simple relationship between prepro-ET-1 mRNA levels and ET-1 accumulation may not be expected, since ET-1 accumulation is determined by a number of processes, including translation into the precursor protein, its processing to big-ET-1, release of big-ET-1, conversion to ET-1 and finally degradation of the active peptide. Since the concentrations of ET-1 accumulating in presence of exogenous big-ET-1 were about 40-fold higher (Figure 7) than those observed in media from cells stimulated with TGF-β (Figure 6), limited ET converting enzyme activity can be excluded. Limited translation and/or limited processing of the precursor protein may account for the non-linear relationship between mRNA levels and accumulation of the active peptide.
At present, two ET receptors, ET-A and ET-B, have been identified; and mRNA for both receptors was found to be expressed in MRC-5 and phLFb. ET-B receptor mRNA appears to be strongly regulated, as indicated by the marked down-regulation by TGF-β, whereas expression of ET-A receptors appears to be constitutive (Figure 8B and C). Constitutive expression of ET-A receptors, but strong regulation of ET-B receptor expression has also been described for other tissues, for example rat mesenteric artery (Uddman et al., 2002).
To check whether ET receptors in human lung fibroblasts are functional, ET-1-induced cellular DMR was measured in MRC-5 cells. DMR allows label-free real-time analysis of signalling pathway activation by GPCRs in living cells (Antony et al., 2009; Schröder et al., 2010). The direction of G-protein pathway mediated DMR depends on the cell-type (Schröder et al., 2010), and we recently showed that in MRC-5 cells β2-adrenoceptor agonists, like direct activation of AC by forskolin, induced negative DMR (Lamyel et al., 2011). In the present experiments, ET-1 induced in a concentration-dependent manner a positive DMR. The non-selective ET receptor antagonist bosentan inhibited ET-1-induced DMR in a manner suggesting a competitive interaction. Combination of the ET-A and ET–B receptor selective antagonists BQ123 and BQ788, in concentrations, in which they may selectively block the respective receptor (Ishikawa et al., 1994), inhibited the ET-1-induced DMR much more effectively than each antagonist alone. This indicates that in MRC-5 human lung fibroblasts both ET receptors are functional. BQ788 alone had only minor effects indicating that ET-A receptors alone are able to induce almost the full DMR response. However, after blockade of ET-A receptors, ET-B receptors appear to be able to maintain a substantial response.
Bosentan attenuated the up-regulation of prepro-ET-1 expression and ET-1 accumulation induced by sub-maximal concentrations of TGF-β (Figures 5 and 6B), indicating that ET-1 may contribute in a kind of auto-regulatory positive feedback to the regulation of its own expression and release. This effect appears to be mediated via ET-A receptors, since only BQ123, but not BQ788 mimicked the effect of bosentan. A similar ET-A receptor-mediated feedback mechanism was also described for the TNF-α-induced up-regulation of ET-1 in human airway smooth muscle cells (Knobloch et al., 2009).
In both, MRC-5 cells and phLFb, ET-1 exerted proliferative effects as measured by [3H]-thymidine incorporation. Under standard culture conditions (Figure 10A–C), ET-1 induced only a minor proliferative effect. However, bosentan caused a marked inhibition, indicating that endogenously released ET-1 exerts already a significant proliferative signal. The inhibitory effect of bosentan was surmountable by exogenous ET-1, indicating a competitive interaction between ET-1 and bosentan, and supporting that the effect of bosentan was caused by specific ET receptor blockade. As only BQ123, but not BQ788, mimicked the effect of bosentan, it is concluded that ET-A receptors mediate the proliferative stimulus. In phLFb, the inhibitory effect of bosentan was smaller, but on the other side ET-1 exerted a stronger proliferative effect. These functional observations are in line with lower expression levels of ET-1 in phLFb compared with MRC-5 cells. ET-1 mediated stronger proliferative effects, when applied already at the time of cell dissemination, presumably a condition with a lower endogenous ET-1 tone and in line with this conclusion, the potency of exogenous ET-1 was also higher. Under these conditions, a proliferative effect of ET-1 was confirmed by cell count. In contrast to present observations, Préfontaine et al. (2008) reported that ET-A and ET-B receptor were involved in ET-1-induced proliferation in rat lung fibroblasts, whereas Gallelli et al. (2005) showed in line with the present data that in phLFb only ET-A receptors mediate proliferative effects. Thus, species difference in the role of ET-A and ET-B receptors must be considered.
Endogenously released ET-1 appears to drive also myofibroblast differentiation as bosentan inhibited α-smooth muscle actin expression. However, ET-1, although up-regulated by exposure to TGF-β, appears not to be involved in TGF-β-induced myofibroblast differentiation, as bosentan did not affect TGF-β-induced up-regulation of α-smooth muscle actin expression (Figure 11).
Furthermore, ET-1 stimulated also collagen synthesis as measured by [3H]-proline incorporation in MRC-5 cells and phLFb. In agreement with the already discussed higher endogenous endothelinergic tone in MRC-5 cells compared to phLFb, bosentan and the ET-A receptor antagonist BQ123 caused a significant reduction of [3H]-proline incorporation in MRC-5 cells, but not in phLFb. Nonetheless, in both cell types, bosentan and BQ123 effectively antagonized the effect of ET-1 in a surmountable manner, whereas BQ788 was ineffective. Direct determination of collagen by the QuickZyme collagen assay confirmed the marked stimulatory effect of ET-1 on collagen synthesis. The observation, that ET-1 up-regulated the mRNA of collagen I-α1, a major collagen type in lung fibroblasts, indicates that enhanced collagen gene transcription may at least contribute to the up-regulation of collagen synthesis.
Activation of ERK–MAPK has been shown to be crucially involved in muscarinic receptor mediated stimulation of proliferation (Matthiesen et al., 2007) and collagen synthesis (Haag et al., 2008) in MRC-5 cell. In the present experiments, ET-1, like oxotremorine, induced a rapid, but transient activation of the ERK-MAPK (Figure 15A). In agreement with previous findings that muscarinic receptors in human lung fibroblasts are Gi coupled (Matthiesen et al., 2006; 2007; Haag et al., 2008), pertussis toxin prevented oxotremorine-induced ERK activation. It inhibited, however, only partially activation of ERK caused by ET-1 (Figure 15), indicating that ET-1-induced ERK activation may be mediated in part via Gi, but to a large extent also via pertussis-insensitive G-proteins, possible Gq or G11/12. In correspondence, pertussis toxin prevented muscarinic stimulation of collagen synthesis, but inhibited only partially ET-1-induced collagen synthesis (Figure 15A). Nonetheless, inhibition of ERK MAP kinase pathway by PD 098059 abolished ET-1-induced stimulation of collagen synthesis (Figure 16B), proving that this pathway is crucially involved not only in proliferative effects of ET-1 (Gallelli et al., 2005) but also in its effects on collagen synthesis.
In conclusion, human lung fibroblasts express a functional autocrine/paracrine endothelinergic system, which may, in interaction with other mediators such as TGF-β, drive pro-fibrotic features. Thus, ET-1 is highly up-regulated by TGF-β and can mediate stimulatory effects on proliferation, myofibroblast differentiation and collagen synthesis. Although ET-B receptors are expressed in human lung fibroblasts and their functional significance could be demonstrated by DMR, all other so far studied effects of ET-1 appear to be mediated exclusively by ET-A receptor activation.