Cheng-Lun Na

Altered stability of pulmonary surfactant in SP-C-deficient mice

The surfactant protein C (SP-C) gene encodes an extremely hydrophobic, 4-kDa peptide produced by alveolar epithelial cells in the lung. To discern the role of SP-C in lung function, SP-C-deficient (−/−) mice were produced. The SP-C (−/−) mice were viable at birth and grew normally to adulthood without apparent pulmonary abnormalities. SP-C mRNA was not detected in the lungs of SP-C (−/−) mice, nor was mature SP-C protein detected by Western blot of alveolar lavage from SP-C (−/−) mice. The levels of the other surfactant proteins (A, B, D) in alveolar lavage were comparable to those in wild-type mice. Surfactant pool sizes, surfactant synthesis, and lung morphology were similar in SP-C (−/−) and SP-C (+/+) mice. Lamellar bodies were present in SP-C (−/−) type II cells, and tubular myelin was present in the alveolar lumen. Lung mechanics studies demonstrated abnormalities in lung hysteresivity (a term used to reflect the mechanical coupling between energy dissipative forces and tissue-elastic properties) at low, positive-end, expiratory pressures. The stability of captive bubbles with surfactant from the SP-C (−/−) mice was decreased significantly, indicating that SP-C plays a role in the stabilization of surfactant at low lung volumes, a condition that may accompany respiratory distress syndrome in infants and adults.

Biogenesis of lamellar bodies, lysosome-related organelles involved in storage and secretion of pulmonary surfactant.

Lamellar bodies are members of a subclass of lysosome-related organelles referred to as secretory lysosomes. The principal constituents of the lamellar body, surfactant phospholipids, are organized into tightly packed, bilayer membranes in a process that is strongly influenced by the lung-specific, hydrophobic peptide SP-B. Newly synthesized SP-B is transported from the Golgi to the lamellar body via multivesicular bodies; in contrast, preliminary evidence suggests that newly synthesized surfactant phospholipids are transported from the ER and incorporated into the internal membranes of the lamellar body via a distinct pathway.

Adaptation and increased susceptibility to infection associated with constitutive expression of misfolded SP-C.

Mutations in the gene encoding SP-C (surfactant protein C; SFTPC) have been linked to interstitial lung disease (ILD) in children and adults. Expression of the index mutation, SP-CΔexon4, in transiently transfected cells and type II cells of transgenic mice resulted in misfolding of the proprotein, activation of endoplasmic reticulum (ER) stress pathways, and cytotoxicity. In this study, we show that stably transfected cells adapted to chronic ER stress imposed by the constitutive expression of SP-CΔexon4 via an NF-κB–dependent pathway. However, the infection of cells expressing SP-CΔexon4 with respiratory syncytial virus resulted in significantly enhanced cytotoxicity associated with accumulation of the mutant proprotein, pronounced activation of the unfolded protein response, and cell death. Adaptation to chronic ER stress imposed by misfolded SP-C was associated with increased susceptibility to viral-induced cell death. The wide variability in the age of onset of ILD in patients with SFTPC mutations may be related to environmental insults that ultimately overwhelm the homeostatic cytoprotective response.

Nedd4-2–Mediated Ubiquitination Facilitates Processing of Surfactant Protein–C

We previously proposed a model of surfactant protein (SP)-C biosynthesis in which internalization of the proprotein from the limiting membrane of the multivesicular body to internal vesicles represents a key step in the processing and secretion of SP-C. To test this hypothesis, alanine mutagenesis of the N-terminal propeptide of SP-C was performed. Adenoviruses encoding mutant proproteins were infected into type II cells isolated from Sftpc(-/-) mice, and media analyzed for secreted SP-C 24 hours after infection. Mutation of S(12)PPDYS(17) completely blocked secretion of SP-C. PPDY (PY motif) has previously been shown to bind WW domains of neural precursor cell-expressed developmentally down-regulated (Nedd) 4-like E3 ubiquitin ligases. Purified recombinant glutathione S-transferase-SP-C propeptide (residues 1-35) bound recombinant Nedd4-2 strongly, and Nedd4 weakly; the S(12)PPDYS(17)mutation abrogated binding of SP-C to Nedd4-2. Immobilized recombinant Nedd4-2 WW domain captured SP-C proprotein from mouse type II cell lysates; in the reverse pulldown, endogenous SP-C in type II cells was captured by recombinant Nedd4-2. To determine if the interaction of Nedd4-2 and SP-C resulted in ubiquitination, the SP-C proprotein was immunoprecipitated from transiently transfected human embryonic kidney 293 cells, and analyzed by SDS-PAGE/Western blotting with ubiquitin antibody. Two ubiquitinated forms of SP-C were detected; ubiquitination was blocked by mutation of K6, but not K34, in the SP-C propeptide. Mutation of K6 also inhibited processing of SP-C proprotein to the mature peptide in human embryonic kidney 293 cells. Nedd4-2-mediated ubiquitination regulates lumenal relocation of SP-C, leading to processing and, ultimately, secretion of SP-C.

Reserve Autophagic Capacity in Alveolar Epithelia Provides a Replicative Niche for Influenza A Virus

Autophagy contributes to cellular homeostasis through metabolite recycling and degradation of cytotoxic protein aggregates and damaged organelles. Although recent studies have established that the requirement for basal autophagy is largely tissue specific, the importance of autophagy for alveolar epithelial cell homeostasis remains an important knowledge gap. In the present study we generated two mouse models, with > 90% or > 50% recombination at the Atg5 locus in the distal respiratory epithelium, to assess the effect of dose-dependent decreases in autophagy on alveolar homeostasis. A 90% decrease in autophagy was well tolerated in young adult mice but resulted in alveolar septal thickening and altered lung mechanics in aged animals, consistent with accumulation of damage over time. By comparison, a 50% decrease in autophagy had no effect on alveolar structure or function throughout the murine life span, indicating that basal autophagy in this compartment exceeds that required for homeostasis. A 50% decrease in autophagy in the bronchoalveolar epithelium significantly attenuated influenza A/H3N2 viral replication, leading to improved lung structure and function and reduced morbidity and mortality after infection. The reserve of autophagic capacity in the alveolar epithelium may provide a niche for replication of influenza A virus.

Deficiency of the BiP cochaperone ERdj4 causes constitutive endoplasmic reticulum stress and metabolic defects.

The BiP cochaperone ERdj4 removes misfolded proteins from the ER lumen by associating with ERAD machinery. Global deficiency of ERdj4 results in widespread constitutive ER stress, decreased survival, and metabolic derangements in mice. These findings indicate that the chaperone activity of ERdj4 is important for ER homeostasis in vivo.

Meckel-Gruber Syndrome Protein MKS3 Is Required for Endoplasmic Reticulum-associated Degradation of Surfactant Protein C

Autosomal dominant mutations in the SFTPC gene are associated with idiopathic pulmonary fibrosis, a progressive lethal interstitial lung disease. Mutations that cause misfolding of the encoded proprotein surfactant protein C (SP-C) trigger endoplasmic reticulum (ER)-associated degradation, a pathway that segregates terminally misfolded substrate for retrotranslocation to the cytosol and degradation by proteasome. Microarray screens for genes involved in SP-C ER-associated degradation identified MKS3/TMEM67, a locus previously linked to the ciliopathy Meckel-Gruber syndrome. In this study, MKS3 was identified as a membrane glycoprotein predominantly localized to the ER. Expression of MKS3 was up-regulated by genetic or pharmacological inducers of ER stress. The ER lumenal domain of MKS3 interacted with a complex that included mutant SP-C and associated chaperones, whereas the region predicted to encode the transmembrane domains of MKS3 interacted with cytosolic p97. Deletion of the transmembrane and cytosolic domains abrogated interaction of MKS3 with p97 and resulted in accumulation of mutant SP-C proprotein; knockdown of MKS3 also inhibited degradation of mutant SP-C. These results support a model in which MKS3 links the ER lumenal quality control machinery with the cytosolic degradation apparatus.

The Phosphatidylcholine Transfer Protein Stard7 is Required for Mitochondrial and Epithelial Cell Homeostasis

Mitochondria synthesize select phospholipids but lack the machinery for synthesis of the most abundant mitochondrial phospholipid, phosphatidylcholine (PC). Although the phospholipid transfer protein Stard7 promotes uptake of PC by mitochondria, the importance of this pathway for mitochondrial and cellular homeostasis represents a significant knowledge gap. Haploinsufficiency for Stard7 is associated with significant exacerbation of allergic airway disease in mice, including an increase in epithelial barrier permeability. To test the hypothesis that Stard7 deficiency leads to altered barrier structure/function downstream of mitochondrial dysfunction, Stard7 expression was knocked down in a bronchiolar epithelial cell line (BEAS-2B) and specifically deleted in lung epithelial cells of mice (Stard7epi∆/∆). Stard7 deficiency was associated with altered mitochondrial size and membrane organization both in vitro and in vivo. Altered mitochondrial structure was accompanied by disruption of mitochondrial homeostasis, including decreased aerobic respiration, increased oxidant stress, and mitochondrial DNA damage that, in turn, was linked to altered barrier integrity and function. Both mitochondrial and barrier defects were largely corrected by targeting Stard7 to mitochondria or treating epithelial cells with a mitochondrial-targeted antioxidant. These studies suggest that Stard7-mediated transfer of PC is crucial for mitochondrial homeostasis and that mitochondrial dysfunction contributes to altered barrier permeability in Stard7-deficient mice.

Airway Epithelial KIF3A Regulates Th2 Responses to Aeroallergens

KIF3A, the gene encoding kinesin family member 3A, is a susceptibility gene locus associated with asthma; however, mechanisms by which KIF3A might influence the pathogenesis of the disorder are unknown. In this study, we deleted the mouse Kif3a gene in airway epithelial cells. Both homozygous and heterozygous Kif3a gene–deleted mice were highly susceptible to aeroallergens from Aspergillus fumigatus and the house dust mite, resulting in an asthma-like pathology characterized by increased goblet cell metaplasia, airway hyperresponsiveness, and Th2-mediated inflammation. Deletion of the Kif3a gene increased the severity of pulmonary eosinophilic inflammation and expression of cytokines (Il-4, Il-13, and Il-17a) and chemokine (Ccl11) RNAs following pulmonary exposure to Aspergillus extract. Inhibition of Kif3a disrupted the structure of motile cilia and impaired mucociliary clearance, barrier function, and epithelial repair, demonstrating additional mechanisms by which deficiency of KIF3A in respiratory epithelial cells contributes to pulmonary pathology. Airway epithelial KIF3A suppresses Th2 pulmonary inflammation and airway hyperresponsiveness following aeroallergen exposure, implicating epithelial microtubular functions in the pathogenesis of Th2-mediated lung pathology.

EMC3 coordinates surfactant protein and lipid homeostasis required for respiration

Adaptation to respiration at birth depends upon the synthesis of pulmonary surfactant, a lipid-protein complex that reduces surface tension at the air-liquid interface in the alveoli and prevents lung collapse during the ventilatory cycle. Herein, we demonstrated that the gene encoding a subunit of the endoplasmic reticulum membrane complex, EMC3, also known as TMEM111 (Emc3/Tmem111), was required for murine pulmonary surfactant synthesis and lung function at birth. Conditional deletion of Emc3 in murine embryonic lung epithelial cells disrupted the synthesis and packaging of surfactant lipids and proteins, impaired the formation of lamellar bodies, and induced the unfolded protein response in alveolar type 2 (AT2) cells. EMC3 was essential for the processing and routing of surfactant proteins, SP-B and SP-C, and the biogenesis of the phospholipid transport protein ABCA3. Transcriptomic, lipidomic, and proteomic analyses demonstrated that EMC3 coordinates the assembly of lipids and proteins in AT2 cells that is necessary for surfactant synthesis and function at birth.