Histone modifications are responsible for decreased Fas expression and apoptosis resistance in fibrotic lung fibroblasts

Although the recruitment of fibroblasts to areas of injury is critical for wound healing, their subsequent apoptosis is necessary in order to prevent excessive scarring. Fibroproliferative diseases, such as pulmonary fibrosis, are often characterized by fibroblast resistance to apoptosis, but the mechanism(s) for this resistance remains elusive. Here, we employed a murine model of pulmonary fibrosis and cells from patients with idiopathic pulmonary fibrosis (IPF) to explore epigenetic mechanisms that may be responsible for the decreased expression of Fas, a cell surface death receptor whose expression has been observed to be decreased in pulmonary fibrosis. Murine pulmonary fibrosis was elicited by intratracheal injection of bleomycin. Fibroblasts cultured from bleomycin-treated mice exhibited decreased Fas expression and resistance to Fas-mediated apoptosis compared with cells from saline-treated control mice. Although there were no differences in DNA methylation, the Fas promoter in fibroblasts from bleomycin-treated mice exhibited decreased histone acetylation and increased histone 3 lysine 9 trimethylation (H3K9Me3). This was associated with increased histone deacetylase (HDAC)-2 and HDAC4 expression. Treatment with HDAC inhibitors increased Fas expression and restored susceptibility to Fas-mediated apoptosis. Fibroblasts from patients with IPF likewise exhibited decreased histone acetylation and increased H3K9Me3 at the Fas promoter and increased their expression of Fas in the presence of an HDAC inhibitor. These findings demonstrate the critical role of histone modifications in the development of fibroblast resistance to apoptosis in both a murine model and in patients with pulmonary fibrosis and suggest novel approaches to therapy for progressive fibroproliferative disorders.     

Histone H4 acetylation dynamics determined by stable isotope labeling with amino acids in cell culture and mass spectrometry

This paper describes an integrated approach that couples stable isotope labeling with amino acids in cell culture to acetic acid-urea polyacrylamide gel electrophoresis (AU-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the quantitation and dynamics of histone H4 acetylation. The 697 acute lymphoblastic cell lines were grown in regular medium and in medium in which lysine was substituted with deuterium-labeled lysine. Histone deacetylase (HDAC) activity was inhibited by addition of the HDAC inhibitor depsipeptide to the culture medium for different exposure times. Histones were extracted from cells pooled from unlabeled, untreated cells and from labeled, treated cells, followed by AU-PAGE separation. Gel bands corresponding to different acetylation states of H4 were excised, in-gel digested with trypsin, and analyzed by MALDI-TOF MS. Detailed information was obtained for both the change of histone H4 acetylation specific to the N terminus and the global transformation of H4 from one acetylation state to another following treatment with the HDAC inhibitor depsipeptide. The kinetics of H4 acetylation was also assessed. This study provides a quantitative basis for developing potential therapies by using epigenetic regulation and the developed methodology can be applied to quantitation of change for other histone modifications induced by external stimuli.     

Cell-specific epigenetic regulation of ChM-I gene expression: crosstalk between DNA methylation and histone acetylation

The expression of the chondromodulin-I (ChM-I) gene, a cartilage-specific gene, is regulated by the binding of Sp3 to the core promoter region, which is inhibited by the methylation of CpG in the target genome in the osteogenic lineage, osteosarcoma (OS) cells. The histone tails associated with the hypermethylated promoter region of the ChM-I gene were deacetylated by histone deacetylase 2 (HDAC2) in three ChM-I-negative OS cell lines. Treatment with an HDAC inhibitor induced the binding of Sp3 in one cell line, which became ChM-I-positive. This process was associated with acetylation instead of the dimethylation of histone H3 at lysine 9 (H3-K9) and, surprisingly, the demethylation of the core promoter region. The demethylation was transient, and gradually replaced by methylation after a rapid recovery of histone deacetylaion. These results represent an example of the plasticity of differentiation being regulated by the cell-specific plasticity of epigenetic regulation.     

Histone deacetylase inhibition regulates miR-449a levels in skeletal muscle cells

microRNAs (miRNAs) are small non-coding RNAs that regulate cellular processes by fine-tuning the levels of their target mRNAs. However, the regulatory elements determining cellular miRNA levels are not well studied. Previously, we had described an altered miRNA signature in the skeletal muscle of db/db mice. Here, we sought to explore the role of epigenetic mechanisms in altering these miRNAs. We show that histone deacetylase (HDAC) protein levels and activity are upregulated in the skeletal muscle of diabetic mice. In C2C12 cells, HDAC inhibition using suberoylanilide hydroxamic acid (SAHA) altered the levels of 24 miRNAs: 15 were downregulated and 9 were upregulated. miR-449a, an intronic miRNA localized within the Cdc20b gene, while being downregulated in the skeletal muscle of diabetic mice, was the most highly upregulated during HDAC inhibition. The host gene, Cdc20b, was also significantly upregulated during HDAC inhibition. Bioinformatics analyses identified a common promoter for both Cdc20b and miR-449a that harbors significant histone acetylation marks, suggesting the possibility of regulation by histone acetylation-deacetylation. These observations suggest an inverse correlation between miR-449a levels and HDAC activity, in both SAHA-treated skeletal muscle cells and db/db mice skeletal muscle. Further, in SAHA-treated C2C12 cells, we observed augmented occupancy of acetylated histones on the Cdc20b/miR-449a promoter, which possibly promotes their upregulation. In vivo injection of SAHA to db/db mice significantly restored skeletal muscle miR-449a levels. Our results provide insights into the potential regulatory role of epigenetic histone acetylation of the miR-449a promoter that may regulate its expression in the diabetic skeletal muscle.