In vitro surfactant protein B deficiency inhibits lamellar body formation.
Surfactant protein (SP) B is essential for normal pulmonary surfactant activity and lamellar body genesis in type 2 cells. However, the role of SP-B in lamellar body genesis is poorly understood. We developed an adenovirus vector expressing antisense SP-B as an alternative in vitro model of SP-B deficiency to begin to explore the role of SP-B in lamellar body genesis. RT-PCR analysis revealed that antisense SP-B expression interfered with translation of endogenous SP-B mRNA. Antisense SP-B expression resulted in reliable in vitro reproduction of many features of SP-B deficiency, including absent mature SP-B and decreased lamellar bodies and SP-C. Light and electron microscopy demonstrated significant reductions in lamellar body number. Western blotting revealed a significant reduction in mature 8-kD SP-B protein and decreased mature SP-C. Our data indicate that antisense SP-B can be effectively used to replicate the SP-B-deficient type 2 cell phenotype in vitro, and provides an attractive alternative to transgenic models for the further study of the role of SP-B in lamellar body genesis.
Foster
Cherie D
CD
Department of Pediatrics, University of Pennsylvania School of Medicine, Children's Hospital of Philadelphia, 3516 Civic Center Blvd., Philadelphia, PA 19104-4318, USA.
Zhang
Peggy X
PX
Gonzales
Linda W
LW
Guttentag
Susan H
SH
D000256:Adenoviridae / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005822:Genetic Vectors; D006801:Humans; D008168:Lung / Q000378:metabolism / Q000502:physiology*; D008854:Microscopy, Electron; D008957:Models, Genetic; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D037701:Pulmonary Surfactant-Associated Protein B / Q000172:deficiency*; D037721:Pulmonary Surfactant-Associated Protein C / Q000737:chemistry; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D016376:Oligonucleotides, Antisense; D037701:Pulmonary Surfactant-Associated Protein B; D037721:Pulmonary Surfactant-Associated Protein C; D012333:RNA, Messenger; D012313:RNA