title: Relaxin prevents cardiac fibroblast-myofibroblast transition via notch-1-mediated inhibition of TGF-β/Smad3 signaling.
abstract: The hormone relaxin (RLX) is produced by the heart and has beneficial actions on the cardiovascular system. We previously demonstrated that RLX stimulates mouse neonatal cardiomyocyte growth, suggesting its involvement in endogenous mechanisms of myocardial histogenesis and regeneration. In the present study, we extended the experimentation by evaluating the effects of RLX on primary cultures of neonatal cardiac stromal cells. RLX inhibited TGF-β1-induced fibroblast-myofibroblast transition, as judged by its ability to down-regulate α-smooth muscle actin and type I collagen expression. We also found that the hormone up-regulated metalloprotease (MMP)-2 and MMP-9 expression and downregulated the tissue inhibitor of metalloproteinases (TIMP)-2 in TGF-β1-stimulated cells. Interestingly, the effects of RLX on cardiac fibroblasts involved the activation of Notch-1 pathway. Indeed, Notch-1 expression was significantly decreased in TGF-β1-stimulatedfibroblasts as compared to the unstimulated controls; this reduction was prevented by the addition of RLX to TGF-β1-stimulated cells. Moreover, pharmacological inhibition of endogenous Notch-1 signaling by N-3,5-difluorophenyl acetyl-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), a γ-secretase specific inhibitor, as well as the silencing of Notch-1 ligand, Jagged-1, potentiated TGF-β1-induced myofibroblast differentiation and abrogated the inhibitory effects of RLX. Interestingly, RLX and Notch-1 exerted their inhibitory effects by interfering with TGF-β1 signaling, since the addition of RLX to TGF-β1-stimulated cells caused a significant decrease in Smad3 phosphorylation, a typical downstream event of TGF-β1 receptor activation, while the treatment with a prevented this effect. These data suggest that Notch signaling can down-regulate TGF-β1/Smad3-induced fibroblast-myofibroblast transition and that RLX could exert its well known anti-fibrotic action through the up-regulation of this pathway. In conclusion, the results of the present study beside supporting the role of RLX in the field of cardiac fibrosis, provide novel experimental evidence on the molecular mechanisms underlying its effects.
authors.affiliation: Department of Experimental and Clinical Medicine-Section of Anatomy and Histology, University of Florence, Florence, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Sassoli
authors.forename: Flaminia
authors.initials: F
authors.lastname: Chellini
authors.forename: Alessandro
authors.initials: A
authors.lastname: Pini
authors.forename: Alessia
authors.initials: A
authors.lastname: Tani
authors.forename: Silvia
authors.initials: S
authors.lastname: Nistri
authors.forename: Daniele
authors.initials: D
authors.lastname: Nosi
authors.forename: Sandra
authors.initials: S
authors.lastname: Zecchi-Orlandini
authors.forename: Daniele
authors.initials: D
authors.lastname: Bani
authors.forename: Lucia
authors.initials: L
authors.lastname: Formigli
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D024042:Collagen Type I / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D006801:Humans; D051379:Mice; D009206:Myocardium / Q000166:cytology*; D058628:Myofibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D041681:NIH 3T3 Cells; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D051880:Receptors, Notch / Q000378:metabolism*; D018000:Receptors, Peptide / Q000378:metabolism; D012065:Relaxin / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D051900:Smad3 Protein / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000199:Actins; D024042:Collagen Type I; D043562:Receptors, G-Protein-Coupled; D051880:Receptors, Notch; D018000:Receptors, Peptide; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; C544013:alpha-smooth muscle actin, mouse; C033395:relaxin receptors; D012065:Relaxin