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

• 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
• 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

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的标志（SSC，硬皮病），而逆转 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，具有强烈的临床前证据 支持其作为器官纤维化的有效抗纤维化分子的作用。但是尽管有强有力的证据 动物模型，基于临床试验的基于松弛素的治疗失败，无法降低SSC患者的皮肤纤维化。一 在临床试验中松弛蛋白失败的潜在分子解释是纤维化中的细胞可能不敏感 放松。我们发现，松弛素受体RXFP1的mRNA和蛋白质水平急剧 与对照组相比，SSC患者的肺和肺成纤维细胞减少。纤维化肺的成纤维细胞 在其体外敏感性对像松弛蛋白样激动剂的体外敏感性受损，但RXFP1表达的增强恢复 在这些细胞中放松蛋白敏感性。但是，导致RXFP1下调的分子机制是 未知。我们已经确定了RXFP1基因远端区域的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的增加> 100倍 SSC（<20倍）的成纤维细胞中的增强部分被部分钝化。另外，转录活性区域 组蛋白的修饰以及上游CPG岛的侧面这个新颖的增强剂。因此，我们假设 该远端RXFP1增强子的异常功能与SSC的表观遗传变化相结合 肺成纤维细胞是RXFP1下调的主要驱动力，并且对这些调节元件进行了修改 并且表观遗传事件将上调RXFP1表达，并使SSC成纤维细胞对放松治疗。 我们将对增强剂区域进行精细映射，并确定负责的转录因子（TF） RXFP1的肺成纤维细胞特异性增强（AIM 1）。确定的TF的表达水平将进一步 与对照和SSC肺和成纤维细胞中的内源性RXFP1表达相关。直接结合 将使用EMSA和CHIP测定法对增强剂的TFS进行分析。我们还将确定 增强子驱动肺成纤维细胞特异性RXFP1表达时围绕增强子的表观遗传修饰（AIM 2）。 组蛋白修饰和DNA甲基化状态均将在对照和SSC肺和成纤维细胞中进行分析 使用分子技术，包括我们的新方法ISH-PLA，一种单细胞表观遗传测定允许 检测组织切片中组蛋白的修饰。这项研究的成功将提供一种新颖的方法 通过识别潜在的治疗方法，反转SSC中基于松弛素的抗纤维化治疗的无效性无效 恢复这些患者纤维化肺中RXFP1表达的靶标。