Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human fetal lung epithelial cells in undifferentiated state and after type II cell differentiation induced by DCI treatment

ABSTRACT: Studies of human fetal lung in explant culture and in isolated epithelial cells have demonstrated that both glucocorticoids and cyclic AMP promote differentiated alveolar type II cell phenotype as assessed by ultrastructural morphology and surfactant production. This project profiles changes in gene expression associated with hormone induced differentiation. Undifferentiated human fetal lung (13-20 wk) epithelial cells were cultured in serum-free medium (control) or with dexamethasone/8-Bromo cyclic AMP/isobutylmethylxanthine (DCI) to promote type II cell differentiation. RNA from five sets of experiments (10 samples) was evaluated using the U133A Affymetrix GeneChip set. Experiment Overall Design: Cells were isolated from 13 individual lungs and the cells from each were cultured in the absence (control) and presence of DCI dexamethasone (10 nM)/8-Br-cyclic AMP (0.1 mM)/isobutylxanthine for 72 h. RNA was isolated from the control and treated cells of each prep. For the 5 sets of microarray experiments (10 total chips, 2 for each control and treated pair), RNA from 2 of the cell preps was analysed as individual microarray comparisons. RNA from 11 cell preps was pooled for the other 3 experiments (4, 4, and 3 cell preps) to provide control and DCI-treated RNA pools.

ORGANISM(S): Homo sapiens

SUBMITTER: Philip Ballard

PROVIDER: E-GEOD-3306 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Gene induction during differentiation of human pulmonary type II cells in vitro.

Wade Kelly C KC Guttentag Susan H SH Gonzales Linda W LW Maschhoff Kathryn L KL Gonzales John J Kolla Venkatadri V Singhal Sunil S Ballard Philip L PL

American journal of respiratory cell and molecular biology 20060210 6

Mature alveolar type II cells that produce pulmonary surfactant are essential for adaptation to extrauterine life. We profiled gene expression in human fetal lung epithelial cells cultured in serum-free medium containing dexamethasone and cyclic AMP, a treatment that induces differentiation of type II cells. Microarray analysis identified 388 genes that were induced > 1.5-fold by 72 h of hormone treatment. Induced genes represented all categories of molecular function and subcellular location, w ...[more]

PMID: 16474099

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Project description:Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3’5’cyclic AMP (cAMP)-dependent Protein Kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in Acrodysostosis. How mutations on PDEs and RIα interfere with regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit. PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα:PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: Translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis Step 2) Processivity: Binding of free cAMP from the cytosol at both CNBs of RIα and Step 3) Product (5’AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that trigger subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, (PDE8 T690P) and RIα (T207A) that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and Fluorescence Polarization (FP) reveal that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5’AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5’AMP release from the PDE, T207A greatly slowed release of substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα:PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.

Dataset Information

Transcription profiling of human fetal lung epithelial cells in undifferentiated state and after type II cell differentiation induced by DCI treatment

Publications

Gene induction during differentiation of human pulmonary type II cells in vitro.

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