A novel approach to reverse the ineffectiveness of relaxin-based anti-fibrotic therapy in SSc

扭转基于松弛素的抗纤维化疗法对 SSc 无效的新方法

基本信息

批准号：
9807333
负责人：
Yingze Zhang
金额：
$ 20.66万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9807333
关键词：
Agonist Animal Model Antibodies Automobile Driving Binding Biological Assay Cells Clinical Clinical Management Clinical Trials CpG Islands DNA DNA Methylation Data Databases Detection Development Distal Down-Regulation EMSA Elements Enhancers Epigenetic Process Event Failure Fibroblasts Fibrosis Gene Expression Genes Genetic Transcription Goals Histone Acetylation Histones Hormones Immunoprecipitation Impairment In Vitro Investigation Luciferases Lung Messenger RNA Methods Modification Molecular Organ Patients Proteins Pulmonary Fibrosis Regulatory Element Relaxin Reporter Reporting Role Scleroderma Simian virus 40 Skin Structure of parenchyma of lung Systemic Scleroderma Techniques Tissues Transcriptional Regulation Up-Regulation base chromatin immunoprecipitation data mining histone methylation histone modification idiopathic pulmonary fibrosis inhibitor/antagonist novel novel strategies pre-clinical programs promoter relaxin receptor skin fibrosis skin lesion success therapeutic target transcription factor

项目摘要

ABSTRACT Skin and lung fibrosis are the hallmarks of systemic sclerosis (SSc, scleroderma), and the reversal of fibrosis is a very challenging clinical problem. Relaxin is a circulating hormone with strong preclinical evidence supporting its role as a potent anti-fibrotic molecule for organ fibrosis. But despite the strong evidence from animal models, relaxin-based therapy in clinical trial failed in reducing the skin fibrosis of SSc patients. One potential molecular explanation for the failure of relaxin in clinical trial is that cells in fibrosis may be insensitive to relaxin. We have found that the mRNA and protein levels of relaxin receptor, RXFP1, are dramatically decreased in the lungs and lung fibroblasts of SSc patients compared to controls. Fibroblasts from fibrotic lungs are impaired in their in vitro sensitivity to a relaxin-like agonist, but enhancement of RXFP1 expression restores relaxin sensitivity in these cells. However, the molecular mechanisms leading to RXFP1 down-regulation is unknown. We have identified a lung fibroblast-specific enhancer in the distal region of the RXFP1 gene that results in >100-fold increase of SV40 promoter-driven luciferase activity in control lung fibroblasts and the enhancement is partially blunted in fibroblasts from SSc (<20-fold). In addition, regions of transcriptionally active histone modifications as well as an upstream CpG island flank this novel enhancer. Therefore, we hypothesize that abnormal function of this distal RXFP1 enhancer, in combination with altered epigenetic changes in SSc lung fibroblasts, is a major driver of RXFP1 down-regulation and that modifications of these regulatory elements and epigenetic events will up-regulation of RXFP1 expression and sensitize SSc fibroblasts to relaxin treatment. We will perform fine mapping of the enhancer region and to identify transcription factors (TFs) responsible for the lung fibroblast-specific enhancement of RXFP1 (Aim 1). The expression levels of Identified TFs will be further correlated with endogenous RXFP1 expression in both control and SSc lungs and fibroblasts. Direct binding of the TFs to the enhancer will be analyzed using EMSA and ChIP assays. We will also determine the role of epigenetic modifications surrounding the enhancer in driving lung fibroblast specific RXFP1 expression (Aim 2). Both histone modification and DNA methylation status will be analyzed in control and SSc lungs and fibroblasts using molecular techniques including our novel method ISH-PLA, a single-cell epigenetic assay allowing detection of histone modifications in tissue sections. The success of this study will provide a novel approach to reverse the ineffectiveness of relaxin-based anti-fibrotic treatment in SSc by identifying potential therapeutic targets for restoring RXFP1 expression in fibrotic lungs of these patients.

抽象的皮肤和肺纤维化是系统性硬化症（SSc，硬皮病）的标志，并且纤维化是一个非常具有挑战性的临床问题。松弛素是一种循环激素，具有强有力的临床前证据支持其作为器官纤维化的有效抗纤维化分子的作用。但尽管有强有力的证据在动物模型中，临床试验中基于松弛素的疗法未能减少 SSc 患者的皮肤纤维化。一松弛素在临床试验中失败的潜在分子解释是纤维化细胞可能不敏感放松。我们发现松弛素受体 RXFP1 的 mRNA 和蛋白水平显着提高与对照组相比，SSc 患者的肺部和肺成纤维细胞减少。来自纤维化肺的成纤维细胞体外对松弛素样激动剂的敏感性受损，但 RXFP1 表达的增强可恢复这些细胞的松弛素敏感性。然而，导致 RXFP1 下调的分子机制是未知。我们在 RXFP1 基因的远端区域发现了一个肺成纤维细胞特异性增强子，导致对照肺成纤维细胞中 SV40 启动子驱动的荧光素酶活性增加 >100 倍 SSc 成纤维细胞的增强作用部分减弱（<20 倍）。此外，转录活性区域组蛋白修饰以及上游 CpG 岛位于该新型增强子的侧翼。因此，我们假设该远端 RXFP1 增强子的异常功能与 SSc 中表观遗传变化的改变相结合肺成纤维细胞是 RXFP1 下调的主要驱动因素，并且这些调节元件的修饰表观遗传事件将上调 RXFP1 表达并使 SSc 成纤维细胞对松弛素治疗敏感。我们将对增强子区域进行精细绘图，并确定负责的转录因子 (TF) RXFP1 的肺成纤维细胞特异性增强（目标 1）。已鉴定的 TF 的表达水平将进一步提高与对照和 SSc 肺及成纤维细胞中的内源 RXFP1 表达相关。直接绑定将使用 EMSA 和 ChIP 检测来分析增强子的 TF。我们还将确定以下角色：增强子周围的表观遗传修饰驱动肺成纤维细胞特异性 RXFP1 表达（目标 2）。将在对照和 SSc 肺和成纤维细胞中分析组蛋白修饰和 DNA 甲基化状态使用分子技术，包括我们的新方法 ISH-PLA，这是一种单细胞表观遗传分析，允许检测组织切片中的组蛋白修饰。这项研究的成功将为我们提供一种新的方法通过识别潜在的治疗方法，扭转以松弛素为基础的抗纤维化治疗对 SSc 的无效性恢复这些患者纤维化肺中 RXFP1 表达的目标。