背景:特发性肺纤维化(IPF)是一种长期,进步,和不可逆的肺间质疾病.转化生长因子β-1(TGF-β1)激活Smad家族成员2(Smad2)和Smad3转录因子是IPF发病机理中的关键事件。然而,关于管理Smad2和Smad3蛋白的分子机制仍然缺乏理解。泛素特异性蛋白酶7(USP7)是一种去泛素酶,在调节细胞内的蛋白质稳定性中起着至关重要的作用。然而,其对TGF-β信号通路的调控及其在IPF中的意义仍未被发现。本研究旨在阐明USP7在TGF-β信号通路中的作用,同时探索所涉及的特定分子机制。此外,这项研究旨在评估靶向USP7抑制剂在IPF中的治疗潜力,从而为IPF的诊断和管理提供新的见解。
方法:我们首先通过Westernblot分析检测了在博来霉素(BLM)诱导的肺纤维化小鼠的肺组织中以及在用或不用TGF-β1处理的Beas-2B细胞中USP7的表达。随后,我们探讨了USP7对纤维化过程和TGF-β1信号通路的影响,利用体外和体内研究。最后,我们评估了USP7特异性抑制剂在IPF小鼠模型中的有效性.
结果:在本研究中,发现USP7使Smad2和Smad3去泛素化,因此增加了它们的稳定性并促进TGF-β1诱导的促纤维化蛋白的产生,包括α-平滑肌肌动蛋白(α-SMA)和纤连蛋白1(FN-1)。抑制或敲低USP7导致Smad2和Smad3蛋白水平降低,导致FN-1,I型胶原α1链(Col1A1)的表达降低,TGF-β1在人肺上皮细胞中诱导α-SMA。这些发现表明USP7的过表达减少Smad2/3泛素化,而抑制或敲低USP7增强其泛素化。USP7在IPF肺中大量表达。在博来霉素诱导的肺损伤中,USP7、Smad2和Smad3的表达上调。在博来霉素诱导的肺纤维化模型中,USP7抑制剂P22077降低了FN-1和I型胶原蛋白的表达以及Smad2/3和胶原蛋白在肺组织中的沉积。
结论:本研究表明USP7通过稳定Smad2和Smad3促进TGF-β1信号传导。USP7对IPF进展的贡献表明它可能是可行的治疗靶标。
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a long-term, progressive, and irreversible pulmonary interstitial disease. The activation of Smad family member 2 (Smad2) and
Smad3 transcription factors by transforming growth factor β-1 (TGF-β1) is a critical event in the pathogenesis of IPF. However, there is still a lack of understanding regarding the molecular mechanisms governing Smad2 and
Smad3 proteins. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a vital role in regulating protein stability within cells. However, its regulation of the TGF-β signaling pathway and its significance in IPF remain undiscovered. This study aims to clarify the function of USP7 in the TGF-β signaling pathway, while simultaneously exploring the specific molecular mechanisms involved. Additionally, this study seeks to evaluate the therapeutic potential of targeted USP7 inhibitors in IPF, thereby providing novel insights for the diagnosis and management of IPF.
METHODS: We first detected the expression of USP7 in lung tissues of mice with Bleomycin (BLM)-induced pulmonary fibrosis and in Beas-2B cells treated with or without TGF-β1 through Western blot analysis. Subsequently, we explored the influence of USP7 on fibrotic processes and the TGF-β1 signaling pathway, utilizing in vitro and in vivo studies. Finally, we assessed the effectiveness of USP7-specific inhibitors in an IPF murine model.
RESULTS: In the present study, USP7 was found to de-ubiquitinate Smad2 and
Smad3, consequently increasing their stability and promoting the TGF-β1-induced production of profibrotic proteins including α-smooth muscle actin (α-SMA) and fibronectin 1 (FN-1). Inhibition or knockdown of USP7 resulted in decreased levels of Smad2 and
Smad3 proteins, leading to reduced expression of FN-1, Collagen Type I Alpha 1 Chain (Col1A1), and α-SMA induced by TGF-β1 in human pulmonary epithelial cells. These findings demonstrate that overexpression of USP7 reduces Smad2/3 ubiquitination, whereas inhibition or knockdown of USP7 enhances their ubiquitination. USP7 is abundantly expressed in IPF lungs. The expressions of USP7, Smad2, and
Smad3 were upregulated in bleomycin-induced lung injury. The USP7 inhibitor P22077 reduced the expression of FN-1 and type I collagen as well as Smad2/3 and collagen deposition in lung tissue in a model of pulmonary fibrosis induced by bleomycin.
CONCLUSIONS: This study demonstrates that USP7 promotes TGF-β1 signaling by stabilizing Smad2 and Smad3. The contribution of USP7 to the progression of IPF indicates it may be a viable treatment target.