Surfactant protein A (SP-A) is an excellent marker of fetal lung maturity and type II cell differentiation. The SP- A gene is silent throughout most of gestation;transcription is induced after ~85% of gestation is complete with increased surfactant lipid synthesis. SP-A gene expression in human fetal lung (HFL) is induced by cAMP and inhibited by hypoxia and transforming growth factor-? (TGF-?). To further define genetic and epigenetic mechanisms for type II cell differentiation, developmental and hormonal regulation of SP-A expression, we are investigating the roles of microRNAs (miRNAs, miRs). We observed that members of the miR-200 and miR-29 families were significantly upregulated in mouse and HFL with type II cell differentiation. This was associated with decreased expression of known miR-200 targets, the repressive transcription factors ZEB1 and ZEB2, as well as functionally related factors, Snail1 and Snail2. ZEB1/2 and Snail1/2 are induced by TGF-? and serve critical roles in epithelial-to-mesenchymal transition (EMT), a process that contributes to branching morphogenesis, tumor progression and, possibly, fibrosis. Notably, both miR-200s and miR-29s are suppressed by TGF-? and inhibited in pulmonary fibrosis. We found that overexpression of ZEB1 or ZEB2 in cultured HFL type II cells blocked cAMP stimulation of SP-A expression, while cAMP markedly inhibited ZEB1/2 and Snail1/2. Both miR-29s and miR-200s target TGF-? and stem cell factor, KLF4, while miR-29 targets transcriptional silencing factors, DNA methyltransferases 3a/3b and histone deacetylase (HDAC)4. These findings support the overall hypothesis that miR-200 and miR-29 families and their targets serve important, interacting regulatory roles in chromatin modification, type II cell differentiation and function in fetal lung Moreover, we propose that an imbalance in this miRNA/target relationship can result in impaired alveolarization and lung pathology. To test this thesis, we propose the following specific aims.
In Aim 1, we will complete ongoing studies to analyze temporal and spatial changes in expression of miR-200 and miR-29, ZEB1/2 and Snail1/2 in fetal mouse lung and in mid-gestation HFL explants and cells;effects of cAMP, TGF-? and O2 tension will be analyzed and correlated with expression of markers of type II cell differentiation and EMT. Expression and functional roles of transcription factors that potentially regulate these miRNAs, including GATA3/6, p53, Gli, c-Myc, and TTF-1, their developmental and hormonal regulation also will be analyzed.
In Aim 2, quantitative chromatin immunoprecipitation will be used to analyze developmental changes and effects of TGF-?, cAMP, and O2 tension, overexpression and knockdown of miRNAs and EMT factors on SP-A promoter binding of endogenous histone-modifying enzymes, coregulators and modified histones in mouse and HFL.
In Aim 3, effects of conditional deletion of the miR-200 and miR-29 family in mice on lung branching morphogenesis, alveolar development, type II cell differentiation and EMT will be assessed, as will their impact on lung pathology.
Development of the fetal lung and differentiation of surfactant producing alveolar type II cells are crucial to survival after birth. Herein; we explore a novel an relatively undefined area involving the roles of two developmentally-regulated microRNA families - miR-200 and miR-29 - which exist in a negative feedback loop with TGF-? and inhibit expression of the functionally-related inducers of epithelial-to-mesenchymal transition (EMT); ZEB1/2 and Snail1/2. In this manner; miR-200 and miR-29 family members may act to promote and maintain epithelial cell differentiation and prevent acquisition of mesenchymal properties. Importantly; these miRNAs also potentially target genes and pathways that direct cells away from a pluripotent state toward a differentiated epithelial cell lineage. Moreover; sustained expression of these miRNA families and suppression of their targets also may serve key roles to prevent the differentiated distal lung epithelium from acquiring mesenchymal properties that can result in a number of pathologic conditions.
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