Riboflavin supplementation does not attenuate hyperoxic lung injury in transgenic (spc-mt)hGR mice.

The aims of this study were to test the hypothesis that mice expressing mitochondrially targeted human glutathione reductase (GR) driven by a surfactant protein C promoter ((spc-mt)hGR) are functionally riboflavin deficient and that this deficiency exacerbates hyperoxic lung injury. The authors further hypothesized that dietary supplementation with riboflavin (FADH) will improve the bioactivity of GR, thus enhancing resistance to hyperoxic lung injury. Transgenic (mt-spc)hGR mice and their nontransgenic littermates were fed control or riboflavin-supplemented diets upon weaning. At 6 weeks of age the mice were exposed to either room air (RA) or >95% O(2) for up to 84 hours. GR activities (with and without exogenous FADH) and GR protein levels were measured in lung tissue homogenates. Glutathione (GSH) and glutathione disulfide (GSSG) concentrations were assayed to identify changes in GR activity in vivo. Lung injury was assessed by right lung to body weight ratios and bronchoalveolar lavage protein concentrations. The data showed that enhanced GR activity in the mitochondria of lung type II cells does not protect adult mice from hyperoxic lung injury. Furthermore, the addition of riboflavin to the diets of (spc-mt)hGR mice neither enhances GR activities nor offers protection from hyperoxic lung injury. The results indicated that modulation of mitochondrial GR activity in lung type II cells is not an effective therapy to minimize hyperoxic lung injury.

Effects of maternal retinoic acid administration in a congenital diaphragmatic hernia rabbit model.

Maternal retinoid administration has beneficial effects on lung development in the nitrofen rodent toxic model of congenital diaphragmatic hernia (DH). We wanted to investigate the effects in a surgical model, where the retinoid signaling pathway is not primarily disrupted by the toxic agent. We created DH in fetal rabbits at day 23 of gestation, administrated to the does all trans-retinoic acid (ATRA) or vehicle (VHC) intramuscularly for 8 consecutive days and harvested normal and operated (DH) fetuses at 31 d (n = 7 in each group). Normal lungs exposed to ATRA had increased surfactant protein mRNA levels without change in type II pneumocyte density. There was no measurable effect on lung-to-body weight ratio and airway morphometry by ATRA. In DH lungs (DH/VHC) surfactant protein mRNA levels were increased, as well as the density of type II pneumocytes. When supplemented with ATRA (DH/ATRA) these parameters returned to normal (VHC). Cell proliferation or apoptosis were not influenced by ATRA supplementation. In conclusion, maternal ATRA supplementation does not affect gross anatomic, morphologic or proliferation indices in hypoplastic lungs related to surgically induced DH in rabbit. However, ATRA lowers surfactant protein expression and normalizes type I/II pneumocyte ratio to what is observed in normal lungs.

Nonspecific interstitial pneumonia, alveolar proteinosis, and abnormal proprotein trafficking resulting from a spontaneous mutation in the surfactant protein C gene.

Human surfactant protein C (hSP-C(1-197)) is synthesized as a 197 amino acid proprotein and cleaved to a mature 3.7 kD form. Although interstitial lung disease in patients with mutations of the hSP-C gene is becoming increasingly recognized, the mechanisms linking molecular events with clinical pathogenesis are not fully defined. We describe a full-term infant with respiratory insufficiency associated with a spontaneous heterozygous mutation resulting in a substitution of lysine for glutamic acid at position 66 (= E66K) of the proximal hSP-C COOH flanking propeptide. Lung histology and biochemical studies of the index patient (hSP-C(E66K)) revealed nonspecific interstitial pneumonia, increased alveolar total phospholipid lacking phosphatidylglycerol, and increased surfactant protein A. Localization of proSP-C from lung sections prepared from this patient using immunofluorescence and immunogold electron microscopy revealed abnormal proSP-C staining in endosomal-like vesicles of type II cells distinct from SP-B. To evaluate the effect of the E66K substitution on intracellular trafficking of proSP-C, fusion proteins consisting of enhanced green fluorescent protein (EGFP) and hSP-C(1-197) (wild type) or mutant hSP-C(E66K) were generated and transfected into A549 cells. EGFP/hSP-C(1-197) was expressed within CD-63-positive, EEA-1-negative vesicles, whereas EGFP/hSP-C(E66K) localized to EEA-1 positive vesicles. The E66K substitution is representative of a new class of SP-C mutation associated with interstitial lung disease that is diverted from the normal biosynthetic pathway. We propose that, similar to other storage disorders, lung injury results from induction of a toxic gain of function induced by the mutant product that is subject to genetic modifiers and environmental influences.

Processing of surfactant protein C requires a type II transmembrane topology directed by juxtamembrane positively charged residues.

Surfactant protein C (SP-C) is a lung-specific protein that is synthesized as a 21-kDa integral membrane propeptide (pro-SP-C) and proteolytically processed to a 3.7-kDa secretory product. Previous studies have shown that palmitoylation of pro-SP-C is dependent on two N-terminal juxtamembrane positively charged residues. We hypothesized that these residues influence modification of pro-SP-C by directing transmembrane orientation. Double substitution mutation of these juxtaposed residues from positive to neutral charged species resulted in complete reversal of transmembrane orientation of pro-SP-C and total abrogation of post-translational processing. Mutation of a single residue resulted in mixed orientation. Protein trafficking studies in A549 cells showed that while the double mutant was retained in the endoplasmic reticulum, single mutants produced a mixed pattern of both endoplasmic reticulum (double mutant-like) and vesicular (wild type-like) expression. Our study demonstrates the crucial role juxtamembrane positively charged residues play in establishing membrane topology and their influence on the trafficking and processing of pro-SP-C. Moreover this study provides a likely precedent for a mechanism in disorders associated with mutations in the membrane-flanking region of integral membrane proteins.