Epigenetic regulation of surfactant protein A gene (SP-A) expression in fetal lung reveals a critical role for Suv39h methyltransferases during development and hypoxia

SP-A gene expression is developmentally regulated in fetal lung. Cyclic AMP (cAMP) induction of SP-A expression in human fetal lung type II cells is O(2) dependent and is mediated by increased binding of TTF-1/Nkx2.1 and NF-κB to a critical response element (TBE). This is associated with increased acetylation and decreased methylation of H3K9 at the TBE. Using chromatin immunoprecipitation analysis of fetal lung between 15.5 and 19.0 days of gestation, we observed that the developmental induction of SP-A was associated with increased recruitment of TTF-1, NF-κB, PCAF, and CBP, as well as enhanced acetylation and decreased methylation of histone H3K9 at the TBE. Importantly, expression and TBE binding of the H3K9 methyltransferases, Suv39h1 and Suv39h2, was inversely correlated with the developmental upregulation of SP-A. In human fetal lung epithelial cells, Suv39H1 and Suv39H2 mRNA levels declined with cAMP induction of SP-A. Moreover, hypoxia, which inhibits cAMP stimulation of SP-A, markedly increased Suv39h1 and Suv39h2 binding to the TBE. Finally, short hairpin RNA knockdown of Suv39H1 or Suv39H2 in fetal lung epithelial cells repressed H3K9 methylation and greatly enhanced SP-A expression. Collectively, our findings suggest that Suv39H1 and Suv39H2 are key hypoxia-induced methyltransferases; their decline in fetal lung during late gestation is critical for epigenetic changes resulting in the developmental induction of SP-A.     

Reduction of glucocorticoid receptor ligand binding by the 11-beta hydroxysteroid dehydrogenase type 2 inhibitor, Thiram

Endogenous and synthetic glucocorticoids (GCs), such as cortisol and dexamethasone (Dex), modulate airway inflammation, regulate the production of surfactant by lung epithelial cells, and influence fetal lung maturation. The 11-beta hydroxysteroid dehydrogenase type 2 (HSD2) enzyme catalyzes the oxidation of bioactive cortisol and Dex to their 11-keto metabolites. Thiram (tetramethylthiuram disulfide) specifically inhibits HSD2 activity by oxidizing cysteine residues located in the cofactor binding domain of the enzyme. During studies performed to define a potential role for HSD2 in modulating GC action in human lung epithelial cells, we observed that exposure of intact human lung epithelial cells (NCI-H441) to 50 microM Thiram significantly attenuated the down-stream effects of Dex (100 nM) on the expression of two GC-sensitive genes, pulmonary surfactant proteins A and B. This observation appeared to be inconsistent with simple inhibition of HSD2 activity. Although Thiram inhibited HSD2 oxidase activity in a dose-dependent manner without affecting HSD2 protein expression, Thiram also reduced specific binding of [3H]-Dex to the glucocorticoid receptor (GR). Pre-treatment of cells with 1 mM dithiothreitol (DTT), a thiol-reducing agent, completely blocked the inhibitory effect of Thiram on ligand binding. These results are suggestive that Thiram may alter the ligand-binding domain of the GR by oxidizing critical thiol-containing amino acid residues. Taken collectively, these data demonstrate that attenuated down-stream GC signaling, via decreased binding of ligand to the GR, is a novel cellular effect of Thiram exposure in human lung epithelial cells.     

Evolutionary conservation of an atypical glucocorticoid‐responsive element in the human tyrosine hydroxylase gene

The human tyrosine hydroxylase (hTH) gene has a 42 bp evolutionarily conserved region designated (CR) II at ?7.24 kb, which bears 93% homology to the region we earlier identified as containing the glucocorticoid response element, a 7 bp activator protein‐1 (AP‐1)‐like motif in the rat TH gene. We cloned this hTH‐CRII region upstream of minimal basal hTH promoter in luciferase (Luc) reporter vector, and tested glucocorticoid responsiveness in human cell lines. Dexamethasone (Dex) stimulated Luc activity of hTH‐CRII in HeLa cells, while mifepristone, a glucocorticoid receptor (GR) antagonist, prevented Dex stimulation. Deletion of the 7 bp 5′‐TGACTAA at ?7243 bp completely abolished the Dex‐stimulated Luc activity of hTH‐CRII construct. The AP‐1 agonist, tetradeconoyl‐12,13‐phorbol acetate (TPA), also stimulated hTH promoter activity, and Dex and TPA together further accentuated this response. Chromatin immunoprecipitation assays revealed the presence of both GR and AP‐1 proteins, especially Jun family members, at this hTH promoter site. Dex did not stimulate hTH promoter activity in a catecholaminergic cell line, which had low endogenous GR levels, but did activate the response when GR was expressed exogenously. Thus, our studies have clearly identified a glucocorticoid‐responsive element in a 7 bp AP‐1‐like motif in the promoter region at ?7.24 kb of the human TH gene.