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 bi...