Guidance of pulmonary fibroblast migration during alveolar septal formation

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Destructive parenchymal lung diseases such as emphysema and pulmonary fibrosis are largely irreversible, and strategies for eliciting alveolar repair and regeneration are an important priority. This application addresses mechanisms which guide alveolar fibroblasts to optimal locations for generation of a coherent, mechanically integrated elastic and collagen fiber network. Learning how signaling platforms integrate and condition signals from the extracellular environment during alveolar development is critical for modifying how fibroblasts migrate and transition to myofibroblasts (MF). When their neuropilin 1 (Nrp1) was depleted, pulmonary MF did not diminish but the surrounding alveolar ducts were enlarged. Preliminary studies also showed that collagen enhanced Ras-related C3 botulinum toxin substrate-1 (Rac1) activation, which is required for cell polarization and migration. Hypothesis: Nrp1 and discoidin domain receptor-2 (DDR2) modify PDGFRα-mediated signaling through Rac1 to direct lung fibroblast (LF) migration and extracellular matrix (ECM) remodeling, during alveolar septation. Components of these signaling pathways assemble in membrane lipid rafts (MLR) where integrins link the ECM to the cellular actin cytoskeleton at focal adhesions. In Aim 1, MF from the lungs of mice bearing deletions of PDGFRα or Nrp1 will be used to dissect the signaling pathways which transmit information from collagen, β1-integrins, and DDR2 to activate Rac1 and thereby regulate the formation of lamellipodia. These studies will (a) examine how Nrp1-deletion alters PDGFRα- targeted protein kinases, adapter proteins and guanine-nucleotide exchange factors, (b) evaluate how PDGF-A interacts with Nrp1, and (c) how Nrp1 regulates endosomal trafficking of PDGFRα. Aim 2 will examine defects in collagen fibers of PDGFRα, Nrp1, or DDR2-deleted mice and how these defects impact the positioning of MF and collagen fibers. These studies will show how DDR2 and integrin α2β1 determine the way fibroblasts respond to fibrillar collagen-1, including their polarization of lamellipodia and membrane type-1 matrix metalloproteinase (MT1-MMP) during migration. They will also determine how the rigidity of collagen fibers alters Rac1-activation, focal adhesion formation, and cell migration. Aim 3 will investigate how PDGFRα and Nrp1 interact with DDR2 and, via Rac1, assemble podosomes, where membrane type-1 matrix metalloprotease (MT1-MMP) targets collagen fibers for degradation to direct the migration and positioning of MF. These studies will explore how podosomes and Rac1 enable fibroblasts to probe and remodel collagen fibers along the axis extending into the distal alveolar septum. In all three aims the collagen composition and the rigidity of the cellular environment will be manipulated to define how they influence cell polarity (of lamellipodia and podosomes), Rac1 activation, migration towards stiffer substrates (durotaxis), and the remodeling of collagen fibers. Learning how MLR and their protein constituents integrate and condition signals from the extracellular environment is critical to modifying how fibroblasts migrate and transition to MF. This process is fundamental to understanding these diseases and how they may be remediated through alveolar regeneration. Because currently available treatments do not arrest or reverse alveolar loss, developing strategies for alveolar regeneration could greatly improve clinical outcomes for pulmonary emphysema, which is prevalent in the American veteran population.