Macitentan inhibits the transforming growth factor-β profibrotic action, blocking the signaling mediated by the ETR/TβRI complex in systemic sclerosis dermal fibroblasts

Fibrosis of skin and internal organs is the dominant feature of SSc and, to date, no therapy has been shown to revert or arrest the progression of fibrosis. Myofibroblasts are generally considered to be the major effector cells responsible for the fibrotic events in the disease, since they contribute to the increased synthesis and contraction of the ECM, which is typical of this disorder. In fact, during SSc, FBs may switch from a form that displays a quiescent phenotype toward a proliferating, matrix-producing and contractile phenotype. Although the origin of myofibroblasts is still a matter of debate, recent studies indicate that bone marrow-derived mesenchymal progenitors undergo myofibroblast differentiation in damaged tissues [24] and we recently demonstrated that perivascular cells might contribute to myofibroblast generation under pathologic stimuli [5–7].

In the fibrotic process, TGF-β seems to play a pre-eminent role [25–27]. This molecule exerts its action through the induction (or repression) of many genes that function as downstream targets and contribute to its effects in a coordinated way [28]. Its action is mediated by SMAD family molecule phosphorylation and inhibiting these downstream products blocks TGF-β effects [18]. It has been reported that, in animal model of skin fibrosis, TGF-β was able to induce transcriptional activation of the ET-1 gene in dermal FBs [15], suggesting that these cells possess an active TGF-β/ET-1 axis and both these molecules may modulate fibrogenic responses. Furthermore, their synergistic effect seems to be particularly complex and a co-operation between their own receptors has been hypothesized.

Our results showed that, in skin FBs, ET-1, mirroring the TGF-β behavior, was able to induce myofibroblast activation, as shown by the increased expression of ColA1 and α-SMA. Several reports [20, 29–31] showed that ET-1 induces a profibrotic phenotype in FBs via the increase of ECM protein expression and the decrease of matrix metalloproteinase expression, suggesting that blocking ET-1 signaling by bosentan, a dual ET-1 receptor antagonist, could be a potential therapeutic strategy for fibrotic disorders [29]. The main studies on the anti-fibrotic effect of ETR antagonists on SSc FBs were reported by Shi-Wen et al. [32–35], showing that bosentan may suppress the expression of α-SMA, type I collagen, fibronectin, and CCN2 in SSc lung FBs and that ET-1 acts as a downstream mediator of TGF-β in human lung FBs. Lagares et al. [15] repeated these results in dermal FBs both in in-vitro experiments and in in-vivo animal models. Recently, the ability of MAC to prevent the ET-1 profibrotic effects on SSc FBs has been demonstrated [20]. Thus, ET-1 seems to be strongly involved in the fibrotic process, and blocking its activity by specific receptor antagonists may be helpful in the treatment of fibrotic disorders, although the detailed molecular mechanism of action of these new drugs still needs to be clarified.

In the present work, we showed that ET-1 performs its action sharing the TGF-β signaling pathway. In fact ET-1 stimulation led to SMAD phosphorylation and MAC blocking the ET-1-induced SMAD signaling pathway, preventing the induction of myofibroblast markers. Furthermore, we showed that MAC may affect the ability of TGF-β to induce TβRI phosphorylation. Of note, our data show, for the first time, that MAC, which specifically binds ETRs, also inhibits the ability of TGF-β to induce its direct downstream signaling, resulting in reduced Col1A1 and α-SMA expression.

A possible hypothesis to explain the direct inhibitory effect of MAC on TGF-β signaling is the evidence that TGF-β is able to induce ET-1 production in FBs, and the ability of TGF-β to induce myofibroblast markers is ET-1-dependent [33]. In our experimental setting, MAC, by blocking ET-1 induced after TGF-β stimulation, decreases the expression of both collagen and contractile proteins.

To confirm that the MAC inhibitory effects on TGF-β signaling are mediated by ET-1, we silenced the ET-1 gene in SSc FBs. After silencing, we showed that MAC maintained its inhibiting action on SMAD phosphorylation regardless of ET-1 production, leading us to speculate that the ability of MAC to inhibit TGF-β signaling is independent of ET-1 production. Until some years ago, it was thought that G protein-coupled receptors (GPCRs), like ETRs, and serine/threonine kinase receptors, such as TβRI, along with their respective downstream effectors, represented distinct and linear signaling units that converged on downstream targets. Recently, it has become more clear that GPCR- and serine/threonine kinase receptor-mediated signaling pathways are not mutually exclusive and they may function as partners [36]. The GPCR–receptor tyrosine kinase (RTK) partnerships may result from the activation of RTKs in response to GPCR stimulation [37, 38], thus inducing the formation of GPCR–RTK complexes.

This event has been already described in different biological settings: the platelet-derived growth factor receptor, which is a member of the RTK family, may be found in a tethered complex with the endothelial differentiation gene 1, a member of the GPCR family [39], as well as the insulin receptor (RTK family) and the beta-adrenergic receptors (GPCR family) which may form a functional tethered complex [40, 41].

In our experimental setting, we observed a direct interaction between the ET-1 and the TGF-β receptors, which are members of different receptor families. This interaction includes a physical association, as confirmed by co-immunoprecipitation experiments. Of note, in SSc FBs the co-immunoprecipitated levels were higher than those observed in HC FBs, probably mirroring the evidence that ETARs were significantly higher in SSc FBs. After silencing ETAR, in SSc FB, MAC failed to inhibit the SMAD phosphorylation induced by TGF-β treatment.

We may speculate that this TβRI–ETR complex modulates the effect of ET-1 on the TGF-β specific signaling pathway, leading to the activation of a profibrotic program in FBs. In this scenario, MAC binded in the pocket of the ETR portion of the ET-1/TGF-β receptor complex may induce a conformational change of this complex, responsible for the inhibition of TGF-β signaling. A further speculative and unexplored aspect, in this setting, may be blocking of the TβRI portion of the ET-1/TGF-β receptor complex. However, until now, unlike ET-1, we still do not have any licensed drug able to interfere with TβRI [42], and from a clinical point of view, at present, ET-1 receptor antagonists represent the only possibility we have to modulate this functional complex.

The evidence of this heterogeneous, but functionally active, TβRI–ETR complex suggests the possibility of an antifibrotic effect of MAC, although these preclinical findings need to be translated into the clinical setting. In fact, although many in vitro studies suggested the antifibrotic effect of another ET-1 receptor antagonist, bosentan [29, 32, 33], no clinical benefits were observed when this drug was employed in SSc patients with lung fibrosis [43, 44]. It must be taken into account that the failure of the clinical trials evaluating its efficacy on interstitial lung disease may be biased by the choice of no sensitive primary endpoint, as well as the heterogeneity of the enrolled patients and the lack of histological classifications. On these bases, any further study of ET-1 receptor antagonists in patients with fibrosis might need more stringent inclusion criteria and perhaps use primary and secondary endpoints which might be more responsive to change [45].