Molecular regulation of BMPRII stability in lung fibrosis

摘要 特发性肺纤维化(IPF)是一种慢性的、不可逆转的、与衰老相关的、最终致命的肺部疾病。 肺纤维化患者的中位生存期仅为4-5年。目前还没有逆转纤维化的治疗方法 治愈IPF。抗纤维化治疗药物的发展是IPF治疗中一个尚未得到满足的需求。冷酷无情的 IPF的进展部分是由于纤维化消退失败所致。IPF治疗方法的发展依赖于 对纤维化消解途径的全面认识。越来越多的证据表明， BMPs信号的激活诱导肌成纤维细胞去分化和纤维化消退。关键受体--骨形态发生蛋白 BMPs中的受体II(BMPRII)信号已被证明在纤维化的肺中减少；因此，恢复 肺纤维化的BMPRII是治疗IPF的一种潜在的治疗方法。然而，BMPRII稳定性的分子调控 没有得到很好的研究。在我们的初步数据中，我们发现(I)BMPRII在溶酶体系统中被降解 对转化生长因子-β-1和脂质过氧化诱导剂的反应，它们在肺纤维化的发展中起关键作用； (Ii)Nedd4L稳定BMPRII；(Iii)下调Nedd4L减少BMP4信号，其影响如下 BMPRII过表达挽救；(4)Nedd4L过表达促进肺去分化 肌成纤维细胞。基于这些新的观察结果，我们假设Ned4L促进了BMPRII的稳定性 促进BMPs/BMPRII介导的肌成纤维细胞去分化和肺纤维化的消退。我们 提出三个具体目标来评估我们的假设。首先，我们将通过以下方式确定监管机制 其中Nedd4L稳定BMPRII。我们将确定BMPRII和BMPRII上的泛素化和Nedd4L对接位点 确定Nedd4L介导的K63连接泛素化对BMPRII内化和稳定性的影响。下一首, 我们将确定Ned4L抑制是否在转化生长因子-β-1和脂质过氧化诱导的BMPRII中是必不可少的 退化。最后，我们将确定Nedd4L是否通过稳定 BMPRII和促进BMPs介导的肌成纤维细胞去分化和失活。我们将确定是否 Nedd4L提高BMPRII稳定性促进体外培养肺组织肌成纤维细胞去分化 肌成纤维细胞。一种可诱导的成纤维细胞特异性Nedd4L耗竭小鼠将用于解决和非 化解肺纤维化模型。拟议的研究将解决有关分子的关键知识差距 BMPs/BMPRII对BMPRII稳定性的调节及抗纤维化作用拟议研究的成功将 提示靶向Nedd4L/BMPRII以挽救纤维化肺中BMPRII的表达可能导致新的 阻止肝纤维化进展和促进肝纤维化消退的机会。

Abstract Idiopathic Pulmonary Fibrosis (IPF) is a chronic, irreversible, aging-associated, and ultimately fatal lung disease. The median survival of pulmonary fibrosis patients is only 4-5 years. There is no treatment to reverse fibrosis and cure IPF. Development of anti-fibrotic therapeutics is an unmet need in the treatment of IPF. The relentless progression of IPF is due in part to the failure of fibrosis resolution. The development of therapies for IPF relies on the comprehensive understanding of fibrosis resolution pathways. Accumulating evidence shows that activation of BMPs signaling induces myofibroblast de-differentiation and fibrosis resolution. A key receptor, BMP receptor II (BMPRII), in BMPs signaling has been shown to be reduced in fibrotic lungs; thus, restoration of BMPRII in fibrotic lungs is a potential therapy to treat IPF. However, molecular regulation of BMPRII stability has not been well studied. In our preliminary data, we discovered that (i) BMPRII is degraded in the lysosome system in response to TGF-β1 and lipid peroxidation inducers, which play critical roles in the development of lung fibrosis; (ii) Nedd4L stabilizes BMPRII; (iii) downregulation of Nedd4L reduced BMP4 signaling and the effects were rescued by overexpression of BMPRII; (iv) overexpression of Nedd4L promoted de-differentiation of lung myofibroblasts. Based on these novel observations, we hypothesized that Nedd4L promotes BMPRII stability and facilitates BMPs/BMPRII-mediated myofibroblast de-differentiation and pulmonary fibrosis resolution. We propose three Specific Aims to evaluate our hypothesis. First, we will determine the regulatory mechanisms by which Nedd4L stabilizes BMPRII. We will identify the ubiquitination and Nedd4L docking sites on BMPRII and determine the effect of Nedd4L-mediated K63-linked ubiquitination on BMPRII internalization and stability. Next, we will determine if Nedd4L suppression is essential for TGF-β1- and lipid peroxidation-induced BMPRII degradation. Finally, we will determine if Nedd4L facilitates lung fibrosis resolution through stabilization of BMPRII and promotion of BMPs-mediated myofibroblast de-differentiation and inactivation. We will determine if elevated BMPRII stability by Nedd4L facilitates myofibroblast de-differentiation in ex-vivo cultured lung myofibroblasts. An inducible fibroblast specific Nedd4L depletion mouse will be used in both resolving and non- resolving lung fibrosis models. The proposed studies will address key knowledge gaps regarding molecular regulation of BMPRII stability and anti-fibrotic effects of BMPs/BMPRII. Success of the proposed studies will suggest that targeting Nedd4L/BMPRII to rescue BMPRII expression in fibrotic lungs may lead to new opportunities to halt pro-fibrotic progression and promote fibrosis resolution.