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 (TΒR1), 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, TΒR1 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 TΒR1 promoter and restored TΒR1 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 TΒR1 expression. Blocking the expression of TWIST1 in KFBs could potentially pave a novel therapeutic avenue for keloid treatment.

中文翻译：

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Twist 相关蛋白 1 通过调节肌细胞增强因子 2A 的稳定性促进瘢痕疙瘩成纤维细胞中β受体 1 的转化生长因子


背景 瘢痕疙瘩是由基因异常激活引起的纤维增生性疾病引起的，其潜在机制尚不清楚。碱性螺旋-环-螺旋转录因子 Twist 相关蛋白 1 （TWIST1） 控制组织发育和疾病过程中的细胞增殖和分化。在这项研究中，我们旨在阐明 TWIST1 在瘢痕疙瘩发病机制中的重要作用。方法 应用免疫组化、细胞计数试剂盒-8 测定、western blotting、PCR、基质胶侵袭测定和免疫荧光测定，证明 TWIST1 在正常皮肤和瘢痕疙瘩成纤维细胞中的作用和机制。应用质谱、泛素化测定、染色质免疫沉淀和双荧光素酶报告基因测定来探索 TWIST1 与下游分子的相互作用。结果 在本研究中，我们证实 TWIST1 在患者瘢痕疙瘩组织和瘢痕疙瘩衍生成纤维细胞 （KFBs） 中上调。在体外，TWIST1 抑制阻止了 KFB 增殖、侵袭和激活。我们还发现了 TWIST1 与转化生长因子 β （TGF-β） 信号相关分子 TGF-β 受体 1 （TΒR1）、SMAD 家族成员 2 （Smad2） 和 Smad3 以及 KFB 中平滑肌肌动蛋白、I 型胶原和 III 型胶原纤维化标志α物之间的联系。从机制上讲，我们发现了一种全新的机制，TWIST1 通过该机制与肌细胞增强因子 2A （MEF2A） 相互作用并抑制其泛素化和降解。使用染色质免疫沉淀和双荧光素酶报告基因测定，TΒR1 被确定为 MEF2A 的新下游靶标，直接与其启动子结合。 TWIST1 过表达促进 MEF2A 募集到 TΒR1 启动子，并恢复 TΒR1 功能表达。结论 我们的研究强调了 TWIST1 在瘢痕疙瘩及其相关成纤维细胞发育中的重要功能，部分原因是 MEF2A 依赖性 TΒR1 表达升高。阻断 KFB 中 TWIST1 的表达可能为瘢痕疙瘩治疗铺平一条新的治疗途径。