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Altered epigenetic regulations are involved in fibrotic responses. Epigenetic mechanisms are sensitive to tissue microenvironment changes. Tricarboxylic acid cycle metabolites can directly affect epigenetic states. Idiopathic pulmonary fibrosis (IPF) is a disease with aberrant metabolism, including enhanced glutaminolysis, which converts glutamine to glutamate by glutaminase, GLS-1. IPF fibroblasts are contractile cells with excessive extracellular matrix production, and up-regulated collagen expression. In this study, we investigated the effects of glutamine metabolism on the fibrotic related gene, Col3A1 expression in primary IPF fibroblasts.
Primary IPF lung fibroblasts were obtained from lung transplantation explants or surgical lung biopsies. When cultured fibroblasts reached about 80% confluent in full medium (with 2 mM glutamine), the medium was changed into fresh full glutamine (2 mM) or reduced glutamine (0 and 0.1 mM) for 48 h. In some studies, GLS-1 was silenced by siRNA to inhibit glutaminolysis. Cells were collected to measure both protein and mRNA expression changes of fibrotic-related gene Col3A1. Histone modification changes and related enzymes were examined under the same conditions; the associations between histone modifications and the promoter region of Col3A1 were examined by ChIP assays.
With decreased glutaminolysis, Col3A1 expression is down-regulated in IPF fibroblasts down-regulate mRNA and protein levels. The repressive histone mark H3K27Me3 showed increased levels in reduced glutamine conditions, likely due to the decreased activities of H3K27Me3 demethylases JMJD3/UTX, which requires the glutamine metabolite -KG as a cofactor. ChIP assays demonstrated increased association of Col3A1 promoter region with the repressive mark, H3K27Me3, under glutamine free conditions. With added -KG to the cell culture medium, the Col3A1 expression is recovered at mRNA and protein level, while ChIP assays showed the association with H3K27me3 at its promoter region is decreased.
Our data indicate that fibrotic related genes, such as Col3A1 is epigenetically regulated by glutaminolysis, which is mediated via the α-KG-H3K27Me3 axis. This study demonstrates that altered metabolism regulates gene expression through epigenetic mechanisms, supporting new treatment approaches in lung fibrosis.
Audience Take Away:
Dr. Yan Sanders is an associate professor in Pulmonary Medicine at the University of Alabama at Birmingham. Her research focus is on epigenetic mechanisms in aging and age-related lung diseases. Her background and expertise are in molecular and cell biology, with specific training and focus on epigenetics. Research projects in Dr. Sanders’ lab include exploring epigenetic alterations in tissue injury and repair processes, in diseases such as idiopathic pulmonary fibrosis (IPF); determining the epigenetic mechanisms, specifically histone modifications and DNA methylation, in cellular senescence, aging, and age-related chronic diseases. Dr. Sanders’ group is the first to report epigenetic mechanisms involved in the pathogenesis of IPF. As PI and co-Investigator on NIH or other agencies sponsored projects, Dr. Sanders’ lab has established the fundamentals and in vitro and in vivo models to explore the epigenetic effects of altered nutrition/metabolic state, and aging on lung injury and repair.