Twist-related protein 1 promotes transforming growth factor β receptor 1 in keloid fibroblasts via regulating the stability of myocyte enhancer factor 2A

Abstract

Background: Keloid scarring is caused by a fibroproliferative disorder due to abnormal activation of genes, the underlying mechanism of which is still unclear. The basic helix–loop–helix transcription factor Twist-related protein 1 (TWIST1) controls cell proliferation and differentiation in tissue development and disease processes. In this study, we aimed to clarify the essential role of TWIST1 in the pathogenesis of keloids. Methods: Immunohistochemistry, cell counting kit-8 assays, western blotting, PCR, matrigel invasion assays and immunofluorescence assays were applied to demonstrate the effects and mechanisms of TWIST1 in fibroblasts derived from normal skin and keloids. Mass spectrometry, ubiquitination assays, chromatin immunoprecipitation and dual luciferase reporter assay were applied to explore the interaction of TWIST1 with downstream molecules. Results: In the present study, we confirmed that TWIST1 was upregulated in keloid tissue of patients and in keloid-derived fibroblasts (KFBs). In vitro, TWIST1 inhibition prevented KFB proliferation, invasion and activation. We also discovered a link between TWIST1 and the transforming growth factor β (TGF-β) signaling related molecules TGF-β receptor 1 (TBR1), SMAD family member 2 (Smad2) and Smad3, and the fibrosis markers α-smooth muscle actin, collagen type I and collagen type III in KFBs. Mechanistically, we uncovered a brand-new mechanism by which TWIST1 interacts with myocyte enhancer factor 2A (MEF2A) and suppresses its ubiquitination and degradation. Using chromatin immunoprecipitation and dual-luciferase reporter assay, TBR1 was identified as a novel downstream target of MEF2A, which directly binds to its promoter. Overexpression of TWIST1 promoted the recruitment of MEF2A to the TBR1 promoter and restored TBR1 functional expression. Conclusions: Our research highlights a significant function of TWIST1 in the development of keloid and its related fibroblasts, partially facilitated by elevated MEF2A-dependent TBR1 expression. Blocking the expression of TWIST1 in KFBs could potentially pave a novel therapeutic avenue for keloid treatment.

Keywords

Ubiquitination / Keloid-derived fibroblasts / Twist-related protein 1 / Myocyte enhancer factor 2A / Transforming growth factor β receptor 1 / Transcription factor

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