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.

Distinct effects of surfactant protein A or D deficiency during bacterial infection on the lung.

Mice lacking surfactant protein (SP)-A (SP-A-/-) or SP-D (SP-D-/-) and wild-type mice were infected with group B streptococcus or Haemophilus influenzae by intratracheal instillation. Although decreased killing of group B streptococcus and H. influenzae was observed in SP-A-/- mice but not in SP-D-/- mice, deficiency of either SP-A or SP-D was associated with increased inflammation and inflammatory cell recruitment in the lung after infection. Deficient uptake of bacteria by alveolar macrophages was observed in both SP-A- and SP-D-deficient mice. Isolated alveolar macrophages from SP-A-/- mice generated significantly less, whereas those from SP-D-/- mice generated significantly greater superoxide and hydrogen peroxide compared with wild-type alveolar macrophages. In SP-D-/- mice, bacterial killing was associated with increased lung inflammation, increased oxidant production, and decreased macrophage phagocytosis. In contrast, in the absence of SP-A, bacterial killing was decreased and associated with increased lung inflammation, decreased oxidant production, and decreased macrophage phagocytosis. Increased oxidant production likely contributes to effective bacterial killing in the lungs of SP-D-/- mice. The collectins, SP-A and SP-D, play distinct roles during bacterial infection of the lung.

Human SP-C gene sequences that confer lung epithelium-specific expression in transgenic mice.

We used transgenic mice to identify cis-active regions of the human pulmonary surfactant protein C (SP-C) gene that impart tissue- and cell-specific expression in vivo in the lung. Approximately 3.7 kb of genomic SP-C DNA upstream of the transcription start site was sufficient to direct chloramphenicol acetyltransferase (CAT) reporter gene expression specifically in bronchiolar and alveolar epithelial cells of the lung. To further define cis-active regulatory elements that mediate cell-specific expression, we tested deletions of the parental 3.7-kb human SP-C sequence in transgenic mice. Tissue CAT assays of mice generated with truncations or overlapping internal deletions of the 3.7-kb construct functionally map alveolar cell-specific regulatory elements to within -215 bp of the SP-C promoter. Analysis of SP-C promoter deletions demonstrate that sequences between -3.7 kb and -1.9 kb contain enhancer sequences that stimulate SP-C transgene expression. In situ hybridization studies demonstrate that deletion of the -1,910- to -215-bp region abolishes the ectopic bronchiolar expression seen with the original 3.7-kb SP-C promoter construct. Comparison of sequences from -215 to +1 bp identified consensus binding sites for the homeodomain transcription factor thyroid transcription factor-1 (TTF-1). Cotransfection assays of the human 3.7-kb SP-C or -1,910- to -215-bp SP-C deletion construct with a TTF-1 expression plasmid demonstrates that TTF-1 transactivates the human SP-C gene. These results suggest that the TTF-1 cis-active sites are important in directing cell-specific expression of the SP-C gene in vivo.

Surfactant protein-D regulates surfactant phospholipid homeostasis in vivo.

Surfactant protein D (SP-D) is a 43-kDa member of the collectin family of collagenous lectin domain-containing proteins that is expressed in epithelial cells of the lung. The SP-D gene was targeted by homologous recombination in embryonic stem cells that were used to produce SP-D (+/-) and SP-D (-/-) mice. Both SP-D (-/-) and SP-D (+/-) mice survived normally in the perinatal and postnatal periods. Whereas no abnormalities were observed in SP-D (+/-) mice, alveolar and tissue phosphatidylcholine pool sizes were markedly increased in SP-D (-/-) mice. Increased numbers of large foamy alveolar macrophages and enlarged alveoli were also observed in SP-D (-/-) mice. Phospholipid composition was unaltered in SP-D (-/-) mice, but surfactant morphology was abnormal, consisting of dense phospholipid membranous arrays with decreased tubular myelin. The pulmonary lipoidosis in the SP-D (-/-) mice was not associated with accumulation of surfactant proteins B or C, or their mRNAs, distinguishing the disorder from alveolar proteinosis syndromes. Surfactant protein A mRNA was reduced and, SP-A protein appeared to be reduced in SP-D (-/-) compared with wild type mice. Targeting of the mouse SP-D gene caused accumulation of surfactant lipid and altered phospholipid structures, demonstrating a previously unsuspected role for SP-D in surfactant lipid homeostasis in vivo.

Transcription of the lung-specific surfactant protein C gene is mediated by thyroid transcription factor 1.

Surfactant protein C (SP-C) is expressed in alveolar Type II epithelial cells of the lung. In order to determine the mechanism(s) that regulate gene transcription, we have analyzed the activation of the murine SP-C promoter in mouse lung epithelial cells (MLE cells) and in HeLa cells after co-transfection with a vector expressing rat thyroid transcription factor-1 (TTF-1). TTF-1 transactivated SP-C-chloramphenicol acetyltransferase constructs containing -13 kilobase pairs to -320 base pairs (bp) of the 5 flanking region of the SP-C gene. Essential cis-acting elements were functionally localized to between -320 and -180 bp from the start of transcription by transfection analysis. Five DNase-protected regions, indicating multiple protein-DNA interactions within the -320 bp TTF-1-responsive region of the SP-C gene, were identified by DNase footprint analysis. A 40-bp segment of SP-C DNA from -197 to -158 linked to a heterologous promoter-chloramphenicol acetyltransferase construct activated expression after co-transfection with CMV-TTF-1 in HeLa and MLE cells. The -197 to -158 segment contained two consensus TTF-1 sites, which were specifically identified as TTF-1 binding sites by gel retardation and antibody supershift with MLE cell nuclear extracts and purified TTF-1 homeodomain protein. Site-specific mutagenesis of either of the TTF-1 binding sites completely blocked activation by TTF-1, indicating both sites are required for TTF stimulation of SP-C transcription.

Tumor necrosis factor-alpha increases Mn-SOD expression: protection against oxidant injury.

Antioxidant enzymes, including superoxide dismutase, are important for protecting the lung against O2 injury. Manganese superoxide dismutase (Mn-SOD) is a superoxide anion (O2-.) scavenger located in the mitochondria, a primary site of O2-. production during hyperoxia. We studied the effects of tumor necrosis factor (TNF-alpha), a macrophage-derived cytokine, on Mn-SOD expression in human pulmonary adenocarcinoma cells. TNF-alpha significantly increased Mn-SOD activity and mRNA in a dose-and time-dependent manner. Mn-SOD activity was increased 3-fold and mRNA 20-fold after a 48-h incubation with TNF-alpha (25 ng/ml). To examine the mechanism of this increase, cells were incubated for 48 h with TNF-alpha (25 ng/ml) with or without cycloheximide (10 microns) or actinomycin D (10 micrograms/ml). Actinomycin D blocked the induction of Mn-SOD mRNA by TNF-alpha, but cycloheximide did not. These findings suggest that the effect of TNF-alpha requires gene transcription but not synthesis of new protein intermediates. To test the hypothesis that increased Mn-SOD protects against oxidative injury, pulmonary adenocarcinoma cells were incubated in TNF-alpha (25 ng/ml) for 48 h and then exposed to paraquat (PQ+), an intracellular O2-. generator. Cells pretreated with TNF-alpha had significantly improved survival in PQ+ compared with controls. At the LD50 (6 microns) for control cells, 95% of TNF-alpha-treated cells survived, 85% at the LD75 (10 microns), and 77% at the LD90 (14 microns). Our results suggest that the induction of Mn-SOD by TNF-alpha in pulmonary adenocarcinoma cells is pretranslationally mediated and that increasing Mn-SOD activity with TNF-alpha confers protection against O2 radicals.

Synthesis and processing of the precursor for human mangano-superoxide dismutase.

Superoxide dismutase comprises a family of metalloenzymes that catalyze the oxido-reduction of superoxide anion to H2O2. Manganese superoxide dismutase (Mn-SOD) is encoded by nuclear chromatin, synthesized in the cytosol, and imported posttranslationally into the mitochondrial matrix. We isolated and sequenced complementary DNA encoding human Mn-SOD. The Mn-SOD cDNA was 1001 base pairs long with a single open reading frame. It contained 95 base pairs of 5' untranslated sequence, and 216 base pairs of 3' untranslated sequence, followed by a short polyadenylation tract. The deduced amino acid sequence suggests a mature protein of 198 amino acids preceded by a 24 amino acid leader peptide. A major transcript of 1000 nucleotides was identified by hybridization of the cDNA with RNA isolated from human cells. Precursor Mn-SOD was produced by in vitro transcription of the human Mn-SOD cDNA followed by in vitro translation utilizing rabbit reticulocyte lysate. The primary translation product of the cDNA is a polypeptide of Mr 26,000 as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. When the Mr 26,000 propeptide was incubated with freshly isolated rat liver mitochondria, the peptide was proteolytically processed to a Mr 24,000 polypeptide. Proteolytic processing was accompanied by an energy-dependent import of the peptide into the isolated liver mitochondria. Mature 125I-labelled Mn-SOD, isolated from rabbit liver, was not imported in vitro into mitochondria, indicating that the energy-dependent uptake of Mn-SOD by liver mitochondria was specific for the Mn-SOD precursor.