of inherited deficiency of pulmonary surfactant protein B (SP-B), which results in respiratory failure and a severe form of bronchopulmonary dysplasia (BPD) in infants, suggests a key role of this protein in differentiation of type II cells. Surfactant isolated from SP-B deficient patients has high surface tension and type II cell structure and function is abnormal with failure of lamellar body genesis and incomplete processing of SP-C. We have observed that fetal lung epithelial cells express SP-B mRNA, but unless cultured with glucocorticoid, contain little mature protein and few lamellar bodies. Based on these observations, we hypothesize the expression of mature (8 kDa) SP-B protein is a critical event in differentiation of mature type II cells, promoting fusion of multivesicular bodies to form lamellar bodies where processing of SP-C occurs. The objective of this proposal is to investigate regulatory events in processing of precursor SP-B and SP-C and the role of SP-B protein in surfactant metabolism.
Specific Aim 1 will evaluate the developmental pattern for synthesis and processing of SP-B protein in surfactant metabolism.
Specific Aim 1 will evaluate the developmental pattern for synthesis and processing of SP-B and SP-C proteins and formation of lamellar bodies using fetal rat and human lung explants. The protein studies will use western immunoblotting with epitope specific antibodies as well as pulse-chase immunoprecipitation protocols, and lamellar bodies will be studied with membrane marker antibodies using confocal immunofluorescence and immuno electron microscopy. Studies of Aim 2 will examine responses to modifying levels of SP-B gene expression. SP-B mRNA content will be increased and decreased using recombinant adenoviruses with sense or antisense SP-B cDNA under control of the CMV promoter and hormones will be used to accelerate epithelial cell differentiation. The goal of aim 3 is to study surfactant protein gene expression and phospholipid metabolism in lung tissue from infants with lung disease. SP-B deficient, BPD and control tissue will be cultured as explants for analysis of surfactant protein gene transcription rate and mRNA content, protein processing and content, and synthesis of surfactant phospholipids. Surfactant proteins will also be assayed in lung lavage samples from premature infants to determine whether a developmental deficiency of SP-B contributes to occurrence of respiratory distress and BPD.
Aim 4 will determine the effects of recombinant SP-B expression in lung cells from infants with an inherited deficiency of SP-B. Studies will be carried out in lung explants and with tissue maintained as xenografts in immune deficient mice. The studies of this project will define expression and processing of surfactant proteins during development and enhance our understanding of the role of SP-B in type II cell differentiation and function. Results will relate directly to future development of gene therapy for infants with lung disease due to inherited deficiency of SP-B or other causes.
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