Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose: Twist1 is a member of the tissue-restricted class B of basic-helix-loop-helix (bHLH) transcription factors acting as master regulators on different tissues. Twist1 is controlled by spatial-temporal expression, dimer choice and cellular localization. Twist1 was shown to be a regulator of mesenchymal transition (EMT). Moreover, Twist1 regulates pathways such as Wnt/βcatenin signalling and responses to hypoxia. The aim of the present study was to investigate the role of Twist1 in TGFβ signaling and in fibroblast activation in systemic sclerosis (SSc).
Methods: Twist1 expression was quantified in SSc patients and different mouse models of fibrosis by qPCR, Western Blot and IF. Collagen synthesis was quantified by qPCR and SirCol in fibroblasts overexpressing or lacking Twist1. Interaction of Twist1, E12 and Id was shown by Co-IP. ChIP assays were performed to analyze Twist1transcriptional binding. The role of Twist1 in vivo was evaluated using inducible, conditional knockout mice with either ubiquitinous (Ubc CreER) or fibroblast-specific (col1a2 CreER) depletion of Twist1. Mice were either challenged with bleomycin or infected with constitutively active TGFβ receptor I adenovirus (TBRICA).
Results: Twist1 mRNA was elevated by 330 % in the skin of SSc patients (p = 0.002). Consistently, Twist1 was increased by 490 % (p = 0.008) and by 349 % (p = 0.001) in bleomycin-induced and TBRICA mouse models, respectively. Induction of Twist1 was mediated by TGFβ in a Smad3 dependent manner in vitro and in vivo, as demonstrated using the selective TGFβ receptor I inhibitor SD208 and Smad3/4 siRNA. Twist1 overexpression fostered the pro-fibrotic effects of TGFβ and enhanced fibroblast differentiation and release of collagen. In contrast, fibroblasts lacking Twist1 ameliorated TGFβ signalling. Chronic stimulation with TGFβ upregulated Twist1 and E12 in fibroblasts, but resulted in an even more pronounced induction of Id1 and Id3. Id proteins have great affinity for E12 and compete against class bHLH factor Twist1 for E12 binding. Co-IP revealed a time dependent shift of Twist1/E12 heterodimers to Id/E12 and Twist1/Twist1 complexes. In addition, binding of Twist1 homodimers to the col1a1 and col1a2 promoter was demonstrated using ChIP assay. Mice lacking Twist1 in fibroblasts were less sensitive to bleomycin induced dermal fibrosis with diminished dermal thickening (-69,3 %, p < 0.0001), myofibroblast counts (-62,3 %, p < 0.0001) and hydroxyproline content (-58,1%, p < 0.0001). Mice with ubiquitous Twist1 knockout were protected to a similar degree, highlighting that fibroblasts are the key-effector cells for Twist1 signalling in experimental skin fibrosis. Twist1 deficiency also protected from TBRICA induced dermal fibrosis.
Conclusion: We identified Twist1 as an important downstream mediator of TGFβ in SSc. TGFβ induces Twist1 expression and stimulates its promoter binding, which triggers myofibroblast differentiation and release of collagen. Knockdown of Twist1 ameliorates the pro-fibrotic effects of TGFβ and ameliorates experimental fibrosis in different murine models of SSc. Thus our study characterizes Twist1 as a key-regulator of fibroblast activation in SSc.
Disclosure:
K. Palumbo-Zerr,
None;
A. Liebl,
None;
P. Zerr,
None;
M. Tomcik,
None;
A. Distler,
None;
C. Beyer,
None;
O. Distler,
Sanofi, Active Biotech, Pfizer, Actelion, and Novartis,
2,
Actelion, Sanofi-Aventis, Roche, Genentech, Active Biotec, medac, Novartis, Sinoxa, 4D Science,
5;
G. Schett,
None;
J. H. W. Distler,
None.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/twist1-regulates-transforming-growth-factor-beta-dependent-activation-of-fibroblasts-in-fibrosis/