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Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-β Signaling
Published on: August 3, 2018
Macitentan inhibits the transforming growth factor-β profibrotic action, blocking the signaling mediated by the
Paola Cipriani1, Paola Di Benedetto2, Piero Ruscitti3
1Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L'Aquila, Delta 6 Building, Via dell'Ospedale, 67100, L'Aquila, Italy. paola.cipriani@cc.univaq.it.
Introduction:
Systemic sclerosis (SSc) is a complex and not fully understood autoimmune disease associated with fibrosis of multiple organs. The main effector cells, the myofibroblasts, are collagen-producing cells derived from the activation of resting fibroblasts. This process is regulated by a complex repertoire of profibrotic cytokines, and among them transforming growth factor beta (TGF-β) and endothelin-1 (ET-1) play a major role. In this paper we show that TGF-β and ET-1 receptors co-operate in myofibroblast activation, and macitentan, an ET-1 receptor antagonist binding ET-1 receptors, might interfere with both TGF-β and ET-1 pathways, preventing myofibroblast differentiation.
Methods:
Fibroblasts isolated from healthy controls and SSc patients were treated with TGF-β and ET-1 and successively analyzed for alpha smooth muscle actin (α-SMA) and collagen (Col1A1) expression and for the Sma and Mad Related (SMAD) phosphorylation. We further tested the ability of macitentan to interfere with these process. Furthermore, we silenced ET-1 and endothelin-1 receptor A expression and evaluated the formation of an ET-1/TGF-β receptor complex by immunoprecitation assay.
Results:
We showed myofibroblast activation in SSc fibroblasts assessing the expression of α-SMA and Col1A1, after stimulation with TGF-β and ET-1. Macitentan interfered with both ET-1- and TGF-β-induced fibroblast activation. To explain this unexpected inhibitory effect of macitentan on TGF-β activity, we silenced ET-1 expression on SSc fibroblasts and co-immunoprecipitated these two receptors, showing the formation of an ET-1/TGF-β receptor complex.
Conclusions:
During SSc, ET-1 produced by activated endothelia contributes to myofibroblast activation using TGF-β machinery via an ET-1/TGF-β receptor complex. Macitentan interferes with the profibrotic action of TGF-β, blocking the ET-1 receptor portion of the ET-1/TGF-β receptor complex.
Insights
Systemic sclerosis involves myofibroblast activation. Macitentan, an endothelin-1 receptor antagonist, inhibits this process by interfering with both endothelin-1 and transforming growth factor-beta pathways through a novel receptor complex.
Area of Science:
- Immunology
- Cell Biology
- Pharmacology
Background:
- Systemic sclerosis (SSc) is an autoimmune disease characterized by organ fibrosis.
- Myofibroblasts, activated fibroblasts producing collagen, drive fibrosis in SSc.
- Transforming growth factor-beta (TGF-β) and endothelin-1 (ET-1) are key profibrotic cytokines in SSc.
Purpose of the Study:
- To investigate the cooperative role of TGF-β and ET-1 receptors in myofibroblast activation.
- To determine if macitentan, an ET-1 receptor antagonist, can modulate these pathways.
- To elucidate the mechanism by which macitentan affects TGF-β signaling.
Main Methods:
- Fibroblasts from healthy controls and SSc patients were stimulated with TGF-β and ET-1.
- Assessed alpha smooth muscle actin (α-SMA) and collagen (Col1A1) expression.
- Investigated SMAD phosphorylation and the effect of macitentan.
- Silenced ET-1 and its receptor A, and performed immunoprecipitation to detect receptor complexes.
Main Results:
- SSc fibroblasts exhibited myofibroblast activation upon stimulation with TGF-β and ET-1.
- Macitentan inhibited fibroblast activation induced by both ET-1 and TGF-β.
- Formation of an ET-1/TGF-β receptor complex was identified, explaining macitentan's effect on TGF-β signaling.
Conclusions:
- Endothelin-1 contributes to myofibroblast activation in SSc by interacting with the TGF-β pathway via a shared receptor complex.
- Macitentan disrupts this profibrotic signaling by targeting the ET-1 receptor component of the complex.
- This suggests macitentan has potential therapeutic applications in systemic sclerosis by inhibiting fibrosis.
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