The objective of this proposal is to elaborate the mechanism by which glucocorticoids modulate the synthesis of the 35kDa surfactant associated protein (PSP-A). At first the developmental profile of PSP-A synthesis under the influence of glucocorticoids at the protein and RNA level will be studied using an in vivo system. The effects of various doses of steroid on PSP-A synthesis will be examined. The steroid effect on PSP-A synthesis as a function of gender will also be examined to determine whether the sex of the animal modulates the glucocorticoid effect on PSP-A synthesis. These studies may provide insight as to why male fetuses show a higher incidence of respiratory distress syndrome and respond poorly to glucocorticoid treatment. Towards these goals newly synthesized PSP-A from control and dexamethasone-treated animals will be analyzed by two- dimensional gel electrophoresis. PSP-A primary translation products of RNA from the same animals will be analyzed similarly. RNA levels of PSP-A from the two groups will also be examined directly by Northern blotting using a rat PSP-A cDNA probe. Subsequently, experiments will be carried out to determine whether the increased PSP-A synthesis following glucocorticoid treatment results from an increased number of Type II epithelial cells synthesizing PSP-A or whether higher amounts of PSP-A message are produced per Type II cell. Using biochemical approaches we will address the questions of whether glucocorticoids modulate PSP-A synthesis by acting directly at the transcriptional level. Newly synthesized PSP-A mRNA in isolated nuclei from whole lung tissue will be obtained from control or dexamethasone treated animals and its rate of synthesis will be examined by filter hybridization to rat PSP-A cDNA. The stability of PSP-A mRNA will also be examined by pulse-chase experiments if this appears to be a factor in the regulation of PSP-A synthesis. We will also use the morphological technique of tissue in situ hybridization, to determine directly whether the glucocorticoid enhanced PSP-A synthesis reflects a higher number of Type II cells synthesizing PSP-A or increased accumulation of PSP-A mRNA in Type II cells. The experiments outlined here taken together will contribute significantly to our understanding about how glucocorticoids modulate PSP-A synthesis and accelerate lung maturity.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL038288-03
Application #
3354456
Study Section
Respiratory and Applied Physiology Study Section (RAP)
Project Start
1988-02-01
Project End
1991-01-31
Budget Start
1990-02-01
Budget End
1991-01-31
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02115
Rayani, H H; Gewolb, I H; Floros, J (1999) Glucose decreases steady state mRNA content of hydrophobic surfactant proteins B and C in fetal rat lung explants. Exp Lung Res 25:69-79
Rayani, H H; Wert Jr, J J; Floros, J (1998) Expression and cDNA sequence of helix destabilizing protein (HDP) in rat lung. Exp Lung Res 24:101-18
Stuempfle, K J; Floros, J (1997) Caution is advised when cDNA expression libraries are screened by southwestern methodologies. Biotechniques 22:260-4
Stuempfle, K J; Koptides, M; Quinn, P G et al. (1996) In vitro analysis of rat surfactant protein A gene expression. Am J Physiol 270:L504-16
Stuempfle, K J; Koptides, M; Karinch, A M et al. (1996) Preparation of transcriptionally active nuclear extracts from mammalian tissues. Biotechniques 21:48-50, 52
Rishi, A K; Joyce-Brady, M; Fisher, J et al. (1995) Cloning, characterization, and development expression of a rat lung alveolar type I cell gene in embryonic endodermal and neural derivatives. Dev Biol 167:294-306
Rayani, H H; Warshaw, J B; Floros, J (1995) Dexamethasone enhances surfactant protein gene expression in streptozotocin-induced immature rat lungs. Pediatr Res 38:870-7
Kouretas, D; Karinch, A M; Rishi, A et al. (1993) Conservation analysis of rat and human SP-A gene identifies 5' flanking sequences of rat SP-A that bind rat lung nuclear proteins. Exp Lung Res 19:485-503
Veletza, S V; Nichols, K V; Gross, I et al. (1992) Surfactant protein C: hormonal control of SP-C mRNA levels in vitro. Am J Physiol 262:L684-7
Guttentag, S H; Phelps, D S; Warshaw, J B et al. (1992) Delayed hydrophobic surfactant protein (SP-B, SP-C) expression in fetuses of streptozotocin-treated rats. Am J Respir Cell Mol Biol 7:190-7

Showing the most recent 10 out of 18 publications