The N-terminal segment of pulmonary surfactant lipopeptide SP-C has intrinsic propensity to interact with and perturb phospholipid bilayers.
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In the present study, 13-residue peptides with sequences corresponding to the native N-terminal segment of pulmonary SP-C (surfactant protein C) have been synthesized and their interaction with phospholipid bilayers characterized. The peptides are soluble in aqueous media but associate spontaneously with bilayers composed of either zwitterionic (phosphatidylcholine) or anionic (phosphatidylglycerol) phospholipids. The peptides show higher affinity for anionic than for zwitterionic membranes. Interaction of the peptides with both zwitterionic and anionic membranes promotes phospholipid vesicle aggregation, and leakage of the aqueous content of the vesicles. The lipid-peptide interaction includes a significant hydrophobic component for both zwitterionic and anionic membranes, although the interaction with phosphatidylglycerol bilayers is also electrostatic in nature. The effects of the SP-C N-terminal peptides on the membrane structure are mediated by significant perturbations of the packing order and mobility of phospholipid acyl chain segments deep in the bilayer, as detected by differential scanning calorimetry and spin-label ESR. These results suggest that the N-terminal region of SP-C, even in the absence of acylation, possesses an intrinsic propensity to interact with and perturb phospholipid bilayers, thereby potentially facilitating SP-C promoting bilayer-monolayer transitions at the alveolar spaces. (+info)
Expression of a human surfactant protein C mutation associated with interstitial lung disease disrupts lung development in transgenic mice.
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Surfactant Protein C (SP-C) is a secreted transmembrane protein that is exclusively expressed by alveolar type II epithelial cells of the lung. SP-C associates with surfactant lipids to reduce surface tension within the alveolus, maintaining lung volume at end expiration. Mutations in the gene encoding SP-C (SFTPC) have recently been linked to chronic lung disease in children and adults. The goal of this study was to determine whether a disease-linked mutation in SFTPC causes lung disease in transgenic mice. The SFTPC mutation, designated g.1728 G --> A, results in the deletion of exon4, generating a truncated form of SP-C (SP-C(Deltaexon4)). cDNA encoding SP-C(Deltaexon4) was constitutively expressed in type II epithelial cells of transgenic mice. Viable F0 transgene-positive mice were not generated after two separate rounds of pronuclear injections. Histological analysis of lung tissue harvested from embryonic day 17.5 F0 transgene-positive fetuses revealed that SP-C(Deltaexon4) caused a dose-dependent disruption in branching morphogenesis of the lung associated with epithelial cell cytotoxicity. Transient expression of SP-C(Deltaexon4) in isolated type II epithelial cells or HEK293 cells resulted in incomplete processing of the mutant proprotein, a dose-dependent increase in BiP transcription, trapping of the proprotein in the endoplasmic reticulum, and rapid degradation via a proteasome-dependent pathway. Taken together, these data suggest that the g.1728 G --> A mutation causes misfolding of the SP-C proprotein with subsequent induction of the unfolded protein response and endoplasmic reticulum-associated degradation pathways ultimately resulting in disrupted lung morphogenesis. (+info)
Pulmonary surfactant secretion in briefly cultured mouse type II cells.
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There is little information on the regulation of surfactant secretion in mouse type II cells. We isolated type II cells from C57BL/6 and FVB mice, cultured them overnight, and then examined their response to known surfactant secretagogues. Secretion of phosphatidylcholine, surfactant protein (SP)-B and SP-C was stimulated by terbutaline, 5'-N-ethylcarboxyamidoadenosine (NECA), ATP, UTP, TPA, and ionomycin. Phosphatidylcholine secretion was increased approximately twofold by all agonists in both strains of mice. The response to terbutaline and NECA is the same as in rat type II cells, whereas the response to ATP, UTP, TPA, and ionomycin is considerably less. Secretion of SP-B and SP-C was increased sevenfold by terbutaline and threefold by ATP, effects similar to those in rat type II cells. The response to terbutaline was significantly decreased in type II cells from beta(2)-adrenergic receptor null mice. These data establish that briefly cultured type II cells provide a suitable model for investigation of surfactant secretion in normal and genetically altered mice. (+info)
Nuclear factor I/thyroid transcription factor 1 interactions modulate surfactant protein C transcription.
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Surfactant protein C (SP-C; Sftpc) gene expression is restricted to pulmonary type II epithelial cells. The proximal SP-C promoter region contains critical binding sites for nuclear factor I (NFI) and thyroid transcription factor 1 (TTF-1; also called Nkx2.1). To test the hypothesis that NFI isoforms interact with TTF-1 to differentially regulate SP-C transcription, we performed transient transfection assays in JEG-3 cells, a choriocarcinoma cell line with negligible endogenous NFI or TTF-1 activity. Cotransfection of NFI family members with TTF-1 induced synergistic activation of the SP-C promoter that was further enhanced by p300. TTF-1 directly interacts with the conserved DNA binding and dimerization domain of all NFI family members in coimmunoprecipitation and mammalian two-hybrid experiments. To determine whether SP-C expression is regulated by NFI in vivo, a chimeric fusion protein containing the DNA binding and dimerization domain of NFI-A and the Drosophila engrailed transcriptional repression domain (NFIen) was conditionally expressed in mice under control of a doxycycline-inducible transgene. Induction of NFIen in a subset of type II cells inhibited SP-C gene expression without affecting expression of TTF-1 in doxycycline-treated double-transgenic mice. Taken together, these findings support the hypothesis that NFI family members interact with TTF-1 to regulate type II cell function. (+info)
Helical peptoid mimics of lung surfactant protein C.
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Among the families of peptidomimetic foldamers under development as novel biomaterials and therapeutics, poly-N-substituted glycines (peptoids) with alpha-chiral side chains are of particular interest for their ability to adopt stable, helical secondary structure in organic and aqueous solution. Here, we show that a peptoid 22-mer with a biomimetic sequence of side chains and an amphipathic, helical secondary structure acts as an excellent mimic of surfactant protein C (SP-C), a small protein that plays an important role in surfactant replacement therapy for the treatment of neonatal respiratory distress syndrome. When integrated into a lipid film, the helical peptoid SP mimic captures the essential surface-active behaviors of the natural protein. This work provides an example of how an abiological oligomer that closely mimics both the hydrophobic/polar sequence patterning and the fold of a natural protein can also mimic its biophysical function. (+info)
In vivo exposure to hyperoxia induces DNA damage in a population of alveolar type II epithelial cells.
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It is well established that hyperoxia injures and kills alveolar endothelial and type I epithelial cells of the lung. Although type II epithelial cells remain morphologically intact, it remains unclear whether they are also damaged. DNA integrity was investigated in adult mice whose type II cells were identified by their endogenous expression of pro-surfactant protein C or transgenic expression of enhanced green fluorescent protein. In mice exposed to room air, punctate perinuclear 8-oxoguanine staining was detected in approximately 4% of all alveolar cells and in 30% of type II cells. After 48 or 72 h of hyperoxia, 8-oxoguanine was detected in 11% of all alveolar cells and in >60% of type II cells. 8-Oxoguanine colocalized by confocal microscopy with the mitochondrial transmembrane protein cytochrome oxidase subunit 1. Type II cells isolated from hyperoxic lungs exhibited nuclear DNA strand breaks by comet assay even though they were viable and morphologically indistinguishable from cells isolated from lungs exposed to room air. These data reveal that type II cells exposed to in vivo hyperoxia have oxidized and fragmented DNA. Because type II cells are essential for lung remodeling, our findings raise the possibility that they are proficient in DNA repair. (+info)
Surfactant protein C gene variation in the Finnish population - association with perinatal respiratory disease.
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Surfactant protein C (SP-C) is a small hydrophobic protein component of alveolar surfactant, a lipid-protein complex lining the alveolar surface of the lung. Surfactant deficiency is the main cause of respiratory distress syndrome (RDS) in premature infants. RDS is a major risk factor of a chronic lung disease called bronchopulmonary dysplasia (BPD). The dominant mutations of the SP-C gene have recently been associated with interstitial lung diseases. However, the common genetic variation in the surfactant protein C gene has not been studied in detail. In the present study, the exonic variation of the SP-C gene in the Finnish population (n=472) was defined, and the association of the allelic variants with the susceptibility to RDS and BPD was examined. Conformation-sensitive gel electrophoresis (CSGE) was used to determine the extent of exonic variation in the SP-C gene. Methods of genotyping were generated for three biallelic polymorphisms of the SP-C gene's exons 1, 4 and 5, which encode proSP-C. The frequencies of these polymorphisms were evaluated in a study population consisting of 158 DNA samples from full-term infants. In addition, the linkage disequilibrium between the SP-C alleles was evaluated by haplotype analysis of parent-infant triplets. The role of SP-C gene variation in RDS and in BPD was evaluated in a high-risk population of 245 premature infants. According to the present results, the SP-C polymorphisms were associated with RDS and with very premature birth. The strength of allelic associations differed according to the gender of the premature infants. (+info)
Effects of vascular endothelial growth factor on isolated fetal alveolar type II cells.
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Previous investigations gained from in vivo or lung explant studies suggested that VEGF is an autocrine proliferation and maturation factor for developing alveolar type II cells. The objective of this work was to determine whether VEGF exerted its growth and maturation effects directly on isolated type II cells. These were isolated from 19-day fetal rat lung and cultured in defined medium. The presence of VEGF receptor-2 was assessed in cultured cells at the pre- and posttranslational levels. Recombinant VEGF(165), formerly found to be active on lung explants, failed to enhance type II cell proliferation estimated by thymidine and 5-bromo-2'-deoxy-uridine incorporation. It increased choline incorporation in saturated phosphatidylcholine by 27% but did not increase phospholipid surfactant pool size. VEGF (100 ng/ml) left unchanged the transcript level of surfactant proteins (SP)-A, SP-C, and SP-D but increased SP-B transcripts to four times the control steady-state level. VEGF slightly retarded, but did not prevent, the in vitro transdifferentiation of type II into type I cells, as assessed by immunolabeling of the type I cell marker T1alpha. We conclude that, with the exception of SP-B expression, which appears to be controlled directly, the previously observed effects of this VEGF isoform on type II cells are likely to be exerted indirectly through reciprocal paracrine interactions involving other lung cell types. (+info)