The role of transcription factor MEOX2 in lipofibroblast function during alveolarization

转录因子 MEOX2 在肺泡化过程中脂肪成纤维细胞功能中的作用

• 批准号: 10598463
• 负责人: Matthew Riccetti
• 金额: $ 3.36万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-02 至 2023-11-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10598463
• 关键词: 5' Flanking Region; Adult; Alveolar; Binding; Biological Assay; Biology; Bronchopulmonary Dysplasia; Brown Fat; CRISPR/Cas technology; Cardiac; Cell Differentiation process; Cells; Cellular biology; Chemicals; Chronic; Chronic lung disease; Code; Cues; DNA; Data; Data Set; Development; Developmental Biology; Disease; Disease Progression; Distal; Endothelial Cells; Epithelial Cells; Epithelium; FGF10 gene; Fatty Acids; Fetal Lung; Fibroblasts; Gases; Gene Expression; Gene Expression Profile; Genetic; Genetic Techniques; Genetic Transcription; Goals; Grant; Histologic; Homeobox; Homeostasis; Human; In Vitro; Knock-out; Ligands; Limb Development; Luciferases; Lung; Lung diseases; Measures; Mesenchymal; Mesenchyme; Metformin; Molecular Biology; Molecular Genetics; Mus; Mutate; Natural regeneration; Neonatal; Non-Small-Cell Lung Carcinoma; Nucleic Acid Regulatory Sequences; Organoids; Peroxisome Proliferator-Activated Receptors; Peroxisome Proliferators; Phenotype; Platelet-Derived Growth Factor alpha Receptor; Proliferating; Proteins; Pulmonary Surfactants; RNA; Regulation; Response Elements; Role; Signal Transduction; Site-Directed Mutagenesis; Supporting Cell; System; Testing; Training; Transcript; Transcriptional Regulation; alveolar epithelium; body system; cardiovascular endothelium; career; epithelium regeneration; fibrotic lung disease; genetic approach; genetic signature; genomic locus; idiopathic pulmonary fibrosis; in silico; in vitro Assay; in vivo; lipid biosynthesis; lung development; mouse model; novel; overexpression; paracrine; pharmacologic; postnatal; receptor; repaired; restoration; selective expression; single-cell RNA sequencing; surfactant production; targeted treatment; therapeutic target; transcription factor; transcriptome sequencing; uptake

Project Summary Pulmonary lipofibroblasts (LFs) that reside in the distal lung mesenchyme support alveolar epithelial cell homeostasis and repair in both adult-onset fibrotic lung diseases (such as idiopathic pulmonary fibrosis, IPF) and neonatal lung diseases (such as bronchopulmonary dysplasia, BPD). Specifically, LFs: 1) uptake and transfer fatty acids to alveolar type 2 (AT2) epithelial cells during alveolarization for surfactant production, and 2) provide paracrine ligands such as FGF10 that stimulate alveolar type 1 (AT1) and AT2 proliferation and differentiation. A better understanding of the transcriptional regulation of the pulmonary lipofibroblast phenotype will lead to the development of targeted therapies for BPD and IPF. Transcription factor MEOX2 is a signature gene of the murine developmental lipofibroblast, as identified by publicly available RNA-sequencing datasets. In other organ systems, MEOX2 controls fatty acid uptake in cardiovascular endothelial cells, is necessary for brown fat adipogenesis, and is critical for mesenchymal proliferation and differentiation in early limb development. Although MEOX2 is upregulated in non-small cell lung cancer, the role of MEOX2 in lung development is completely unknown. We predicted that MEOX2 would have a role in lipofibroblast differentiation, so we strove to generate preliminary data on MEOX2’s upstream and downstream function. PPAR signaling is a known activator of lipofibroblast differentiation, so we treated human PDGFRa+ fetal lung fibroblasts (IMR90 cells) with pan-PPAR activator metformin, which induced expression of MEOX2. Additionally, we performed a transcription factor motif search at the human MEOX2 locus and identified a putative peroxisome proliferator response element (PPRE). We then overexpressed MEOX2 in IMR90 cells and determined that MEOX2 is sufficient to induce expression of lipofibroblast signature genes PLIN2 and TCF21. The central hypothesis of this grant is that MEOX2 is directly regulated by PPARA signaling and is necessary and sufficient for establishment of the lipofibroblast lineage and function during alveolarization. Aim 1 will investigate if the putative PPRE is necessary and sufficient for induction of MEOX2 expression through the use of molecular genetic approaches in immortalized human lung fibroblasts. Aim 2 will investigate if MEOX2 is necessary and sufficient to induce lipofibroblast gene expression, fatty acid (FA) uptake and transfer, and support of alveolar epithelial differentiation through 1) in vitro assays combined with transient MEOX2 knockout and overexpression constructs, and 2) an in vivo conditional fibroblast specific MEOX2 knockout during early alveolarization. Our long-term goal is to identify MEOX2’s role as a transcription factor in the development of the lung mesenchyme, with the hope of discovering therapeutic targets for chronic neonatal and adult lung diseases. This training plan combines, molecular, cellular, and developmental biology to answer fundamental questions about pulmonary fibroblast biology. My career goal is to become a leader in understanding how transcription factors regulate cellular differentiation in development, and how they may be targetable for therapies in disease.

项目摘要 肺脂肪纤维细胞（LFS）位于远端肺间质支持肺泡上皮细胞中 两种成年纤维化肺部疾病的稳态和修复（例如特发性肺纤维化，IPF） 和新生儿肺部疾病（例如支气管肺发育不良，BPD）。特别是LFS：1）吸收和 肺泡产生肺泡化期间，将脂肪酸转移到2型肺泡2（AT2）上皮细胞，并将其转移到表面活性剂的产生中，并 2）提供旁分泌配体，例如刺激牙槽1（AT1）和AT2增殖的FGF10，以及 分化。更好地理解肺脂肪纤维细胞表型的转录调节 将导致开发BPD和IPF的靶向疗法。转录因子MEOX2是一个签名 通过公开可用的RNA序列数据集确定的鼠发育性脂肪纤维细胞基因。在 其他器官系统，MEOX2控制心血管内皮细胞中脂肪酸的摄取，对于 棕色脂肪成生成，对于早期肢体的间充质增殖和分化至关重要 发展。尽管MEOX2在非小细胞肺癌中进行了更新，但Meox2在肺中的作用 发展是完全未知的。我们预测MEOX2将在脂肪纤维细胞分化中发挥作用， 因此，我们努力生成有关Meox2上游和下游功能的初步数据。 PPAR信号是 脂肪纤维细胞分化的已知激活剂，因此我们处理了人类PDGFRA+胎儿肺成纤维细胞（IMR90 细胞）用泛铂活化剂二甲双胍，诱导Meox2的表达。此外，我们进行了 转录因子图案搜索人类Meox2基因座，并确定了推定的过氧化物组增殖物 响应元素（PPRE）。然后，我们在IMR90细胞中过表达MEOX2，并确定Meox2是 足以诱导脂肪纤维细胞签名基因PLIN2和TCF21的表达。中心假设 Grant是Meox2直接受PPARA信号传导的调节，并且足以建立 肺泡化期间的脂肪纤维细胞谱系和功能。 AIM 1将调查假定的PPRE是否 通过使用分子遗传方法，必要且足够的诱导Meox2表达 在永生的人肺成纤维细胞中。 AIM 2将调查MEOX2是否需要且足以诱导 脂肪纤维细胞基因表达，脂肪酸（FA）摄取和转移，并支持肺泡上皮 通过1）体外测定与瞬时MEOX2敲除和过表达的分化 构造和2）在早期肺泡化期间体内条件成纤维细胞特异性MEOX2敲除。我们的 长期目标是确定Meox2在肺间隙发展中的转录因子的作用，即 希望发现针对慢性新生儿和成人肺部疾病的治疗靶标。这个培训计划 结合了分子，细胞和发育生物学，以回答有关肺部的基本问题 成纤维细胞生物学。我的职业目标是成为了解转录因素如何调节的领导者 细胞发育的细胞分化，以及它们如何针对疾病的疗法。