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.

项目概要 位于远端肺间质的肺脂肪成纤维细胞（LF）支持肺泡上皮细胞 成人发病的纤维化肺疾病（如特发性肺纤维化，IPF）的稳态和修复 和新生儿肺部疾病（例如支气管肺发育不良，BPD），具体而言，LF：1）摄取和 在肺泡化过程中将脂肪酸转移至肺泡 2 型 (AT2) 上皮细胞以产生表面活性剂，以及 2) 提供旁分泌配体，例如刺激肺泡 1 型 (AT1) 和 AT2 增殖的 FGF10， 更好地了解肺脂肪成纤维细胞表型的转录调控。 将导致 BPD 和 IPF 靶向疗法的开发 转录因子 MEOX2 是一个标志。 小鼠发育性脂肪成纤维细胞的基因，通过公开的 RNA 测序数据集鉴定。 在其他器官系统中，MEOX2 控制心血管内皮细胞的脂肪酸摄取，对于 棕色脂肪脂肪生成，对于早期肢体的间充质增殖和分化至关重要 尽管 MEOX2 在非小细胞肺癌中表达上调，但 MEOX2 在肺癌中的作用。 我们预测 MEOX2 在脂肪成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。 一种已知的脂肪成纤维细胞分化激活剂，因此我们处理了人 PDGFRa+ 胎儿肺成纤维细胞 (IMR90 细胞）与泛 PPAR 激活剂二甲双胍诱导 MEOX2 的表达。 在人类 MEOX2 位点搜索转录因子基序并鉴定出假定的过氧化物酶体增殖子 然后我们在 IMR90 细胞中过表达 MEOX2，并确定 MEOX2 是 足以诱导脂肪成纤维细胞特征基因 PLIN2 和 TCF21 的表达。 授予的是 MEOX2 直接受 PPARA 信号传导调节，并且对于建立来说是必要和充分的 目标 1 将研究假定的 PPRE 是否是肺泡化期间的脂肪成纤维细胞谱系和功能。 通过使用分子遗传学方法诱导 MEOX2 表达是必要和充分的 目标 2 将研究 MEOX2 是否是诱导的必要且充分的。 脂肪成纤维细胞基因表达、脂肪酸 (FA) 摄取和转移以及肺泡上皮的支持 通过 1) 体外测定结合瞬时 MEOX2 敲除和过表达进行分化 构建体，2) 早期肺泡化过程中体内条件性成纤维细胞特异性 MEOX2 敲除。 长期目标是确定 MEOX2 作为转录因子在肺间质发育中的作用， 希望能够发现慢性新生儿和成人肺部疾病的治疗靶点。 结合分子、细胞和发育生物学来回答有关肺的基本问题 我的职业目标是成为了解转录因子如何调节的领导者。 发育中的细胞分化，以及它们如何成为疾病治疗的目标。