Role of Emc3/Tmem111 in Alveolar Type 2 (AT2) Cell Function. Overview: Pulmonary surfactant is a complex mixture of lipids and proteins produced by AT2 cells that is secreted into the alveolar spaces to reduce surface tension and prevent alveolar collapse during ventilation. Lack of pulmonary surfactant leads to respiratory failure in 1) preterm infants and adults with respiratory distress syndrome (RDS/ARDS) and 2) chronic interstitial lung diseases (ILD) caused by mutations in genes encoding surfactant proteins (e.g. ABCA3, SFTPA, SFTPB, and SFTPC). ABCA3, a phospholipid transporter, and SP-B are critical for the formation of lamellar bodies and for surfactant function. Misrouting of mutant SP- C or loss of ABCA3 cause AT2 cell injury leading to ILD. While genetic diagnoses for diseases of surfactant homeostasis are now possible in newborn infants, there are no effective therapies other than lung transplantation for these usually fatal disorders. Lacking is knowledge regarding the specific AT2 cell machinery that integrates the routing and processing of surfactant lipids and proteins in the AT2 cells, and the molecular mechanisms by which disruption of these pathways causes AT2 cell injury, surfactant deficiency and alveolar remodeling. We have identified Emc3/Tmem111 as a critical ER component of the cellular machinery that regulates the processing, routing, and function of SP-B, SP-C, ABCA3, and lipids in AT2 cells. In this project, we will identify the intracellular sites and functions of EMC3 in vivo and in vitro. The effects of loss of function of EMC3 on lung structure, surfactant homeostasis, and lung function will be determined. Its role in a proposed cell-specific ER/EMC3 complex, its protein cargoes, and its requirement for surfactant homeostasis in AT2 cell function will be identified in mouse models in vivo, in primary AT2 cells isolated from the mice, and immortalized airway epithelial cells, representing models of both human and mouse AT2 cell function. Immunofluorescence and confocal microscopy will be used to precisely identify the intracellular sites of action, and an Emc3-flag-tomato construct will be used in conjunction with antibodies for Co-IP experiments to identify protein cargoes by proteomic analysis. Lipidomic analyses and ultrastructural studies will identify its role in lamellar body formation and surfactant lipid homeostasis. The role of EMC3 in ER stress and the recognition, stabilization, and chaperoning of mutant surfactant proteins and ABCA3 produced by defects in SFTPC and ABCA3 genes will be identified. We will determine whether EMC3/EMC complex is regulated by and influences ER stress and endoplasmic reticulum associated degradation (ERAD) pathways that causes AT2 cell toxicity.
Interstitial Lung Diseases (ILDs), represent a diversity of chronic pulmonary disorders that are common causes of morbidity and mortality in children and adults. Alveolar dysfunction, pulmonary fibrosis, and vascular remodeling are associated with chronic ILDs leading to progressive respiratory failure for which they are few effective therapies. Both genetic and environmental factors underlie the pathogenesis of ILDs. Mutations in genes regulating surfactant homeostasis and alveolar type 2 (AT2) cell function, ABCA3, SFTPB, SFTPC, SFTPA, NKX2-1, and Telomerase (and related genes) cause alveolar injury and respiratory failure in neonates, children, and older individuals. We have identified EMC3/TMEM111 as a novel regulator of surfactant homeostasis in AT2 cells, showing that it is (1) required for the chaperoning and routing of surfactant proteins, ABCA3, and surfactant lipids by AT2 cells and is (2) required for lung function at birth. In vivo and in vitro experiments will define the mechanisms by which EMC3 regulates AT2 cell functions. EMC3 is a novel molecular regulator of surfactant homeostasis. Its role in coordinating surfactant lipid and protein trafficking and processing will be identified to further our understanding of the pathogenesis of interstitial lung diseases.
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