O-GlcNAc regulates anti-fibrotic genes in lung fibroblasts through EZH2.

Epigenetic modifications are involved in fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), and contribute to the silencing of anti-fibrotic genes. H3K27me3, a key repressive histone mark, is catalysed by the methyltransferase enhancer of Zeste homologue 2 (EZH2), which is regulated by the post-translational modification, O-linked N-Acetylglucosamine (O-GlcNAc). In this study, we explored the effects of O-GlcNAc and EZH2 on the expression of antifibrotic genes, cyclooxygenase-2 (Cox2) and Heme Oxygenase (Homx1). The expression of Cox2 and Hmox1 was examined in primary IPF or non-IPF lung fibroblasts with or without EZH2 inhibitor EZP6438, O-GlcNAc transferase (OGT) inhibitor (OSMI-1) or O-GlcNAcase (OGA) inhibitor (thiamet G). Non-IPF cells were also subjected to TGF-ß1 with or without OGT inhibition. The reduced expression of Cox2 and Hmox1 in IPF lung fibroblasts is restored by OGT inhibition. In non-IPF fibroblasts, TGF-ß1 treatment reduces Cox2 and Hmox1 expression, which was restored by OGT inhibition. ChIP assays demonstrated that the association of H3K27me3 is reduced at the Cox2 and Hmox1 promoter regions following OGT or EZH2 inhibition. EZH2 levels and stability were decreased by reducing O-GlcNAc. Our study provided a novel mechanism of O-GlcNAc modification in regulating anti-fibrotic genes in lung fibroblasts and in the pathogenesis of IPF.

Epigenetic regulation of IPF fibroblast phenotype by glutaminolysis.

OBJECTIVE: Excessive extra-cellular-matrix production and uncontrolled proliferation of the fibroblasts are characteristics of many fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). The fibroblasts have enhanced glutaminolysis with up-regulated glutaminase, GLS1, which converts glutamine to glutamate. Here, we investigated the role of glutaminolysis and glutaminolysis-derived metabolite α-ketoglutarate (α-KG) on IPF fibroblast phenotype and gene expression. METHODS: Reduced glutamine conditions were carried out either using glutamine-free culture medium or silencing the expression of GLS1 with siRNA, with or without α-KG compensation. Cell phenotype has been characterized under these different conditions, and gene expression profile was examined by RNA-Seq. Specific profibrotic genes (Col3A1 and PLK1) expression were examined by real-time PCR and western blots. The levels of repressive histone H3K27me3, which demethylase activity is affected by glutaminolysis, were examined and H3K27me3 association with promoter region of Col3A1 and PLK1 were checked by ChIP assays. Effects of reduced glutaminolysis on fibrosis markers were checked in an animal model of lung fibrosis. RESULTS: The lack of glutamine in the culture medium alters the profibrotic phenotype of activated fibroblasts. The addition of exogenous and glutaminolysis-derived metabolite α-KG to glutamine-free media barely restores the pro-fibrotic phenotype of activated fibroblasts. Many genes are down-regulated in glutamine-free medium, α-KG supplementation only rescues a limited number of genes. As α-KG is a cofactor for histone demethylases of H3K27me3, the reduced glutaminolysis alters H3K27me3 levels, and enriches H3K27me3 association with Col3A1 and PLK1 promoter region. Adding α-KG in glutamine-free medium depleted H3K27me3 association with Col3A1 promoter region but not that of PLK1. In a murine model of lung fibrosis, mice with reduced glutaminolysis showed markedly reduced fibrotic markers. CONCLUSIONS: This study indicates that glutamine is critical for supporting pro-fibrotic fibroblast phenotype in lung fibrosis, partially through α-KG-dependent and -independent mechanisms, and supports targeting fibroblast metabolism as a therapeutic method for fibrotic diseases.

Modulation of H4K16Ac levels reduces pro-fibrotic gene expression and mitigates lung fibrosis in aged mice.

Histone H4 lysine16 acetylation (H4K16Ac) modulates chromatin structure by serving as a switch from a repressive to a transcriptionally active state. This euchromatin mark is associated with active transcription. In this study, we investigated the effects of H4K16Ac on the expression of pro-fibrotic genes in lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and in an aging murine model of lung fibrosis. Methods: The lung tissues and fibroblasts from human IPF/non-IPF donors and from aged mice with/without bleomycin induced lung fibrosis were used in this study. The H4K16Ac levels were examined by immunohistochemistry or western blots. RNA silencing of H4K16Ac acetyltransferase Mof was used to reduce H4K16Ac levels in IPF fibroblasts. The effects of reduced H4K16Ac on pro-fibrotic gene expression were examined by western blots and real-time PCR. The association of H4K16Ac with these genes' promoter region were evaluated by ChIP assays. The gene expression profile in siRNA Mof transfected IPF cells were determined by RNA-Seq. The impact of H4K16Ac levels on lung fibrosis was evaluated in an aging murine model. Results: Aged mice with bleomycin induced lung fibrosis showed increased H4K16Ac levels. Human lung fibroblasts with siRNA Mof silencing demonstrated reduced H4K16Ac, and significantly down-regulated profibrotic genes, such as α-smooth muscle actin (α-SMA), collagen I, Nox4, and survivin. ChIP assays confirmed the associations of these pro-fibrotic genes' promoter region with H4K16Ac, while in siRNA Mof transfected cells the promoter/H4K16Ac associations were depleted. RNA-seq data demonstrated that Mof knockdown altered gene expression and cellular pathways, including cell damage and repair. In the aging mice model of persistent lung fibrosis, 18-month old mice given intra-nasal siRNA Mof from week 3 to 6 following bleomycin injury showed improved lung architecture, decreased total hydroxyproline content and lower levels of H4K16Ac. Conclusions: These results indicate a critical epigenetic regulatory role for histone H4K16Ac in the pathogenesis of pulmonary fibrosis, which will aid in the development of novel therapeutic strategies for age-related diseases such as IPF.