Pulmonary surfactant maintains the integrity of alveoli by reducing surface tension at the alveolar air-tissue interface. Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of surfactant. Inadequate synthesis of SP-B that occurs in premature infants is associated with the development of newborn respiratory distress syndrome (RDS) and deficiency of SP-B leads to the development of fatal respiratory failure in infants with congenital alveolar proteinosis. Deficiency of SP-B is also found in adult respiratory distress syndrome (ARDS). SP-B mRNA is expressed in a cell type-specific manner in the lung and is developmentally regulated. Glucocorticoids and cAMP induce expression of SP-B mRNA while tumor necrosis factor-alpha (TNF-alpha) inhibits expression of SP- B mRNA. The long-term objectives of this proposal are to elucidate molecular mechanisms that regulate SP-B gene expression.
In Specific Aim 1, transcriptional activators binding to a novel regulatory cis-DNA element in SP-B promoter will be purified by use of sequence-specific DNA affinity chromatography and cDNAs encoding transcriptional activators will be cloned. The cDNAs will be characterized and their role in SP-B promoter function will be analyzed by reconstituted in vitro transcription assay and cotransfection experiments.
In Specific Aim 2, the role of stereospecific alignments between various regulatory cis-DNA elements in SP-B promoter function will be analyzed by altering the helical phasing of individual elements. The role of cooperative interactions between cis-DNA elements and transcriptional activators bound to the DNA elements in SP-B promoter function will be analyzed by in vitro DNA binding studies and cotransfection experiments.
In Specific Aim 3, cis-DNA elements and transcriptional activators that mediate TNF-alpha down regulation of SP-B promoter activity will be identified and mechanisms underlying down regulation of promoter activity will be studied.
In Specific Aim 4, SP-B genomic regions that control cell-specific, developmental and multifactorial regulation of SP-B gene expression in vivo will be identified by development of transgenic mice. The studies described should provide significant new information on molecular mechanisms that control SP-B gene expression, and may aid in the development of newer therapies aimed at prevention and treatment of newborn respiratory distress syndrome.
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