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A5790 - Apoptosis Resistance of IPF Fibroblasts Is Mediated by Glutaminolysis-Dependent Epigenetic Regulation of Pro-Survival Genes
Author Block: Y. Sanders1, L. Bai1, S. Tang1, J. C. Horowitz2, V. J. Thannickal1; 1Medicine, Univ of Alabama At Birmingham, Birmingham, AL, United States, 2University of Michigan Medical School, Ann Arbor, MI, United States.
Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:""Table Normal""; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin-top:0in; mso-para-margin-right:0in; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0in; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:""Calibri"",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} Background: Fibrotic response involves multiple cellular process, including epigenetic changes, which are sensitive to alterations in the tissue microenvironment, such as the flux of tricarboxylic acid (TCA) cycle metabolites. TCA metabolites can directly regulate epigenetic states, in part, by regulating histone modifications. Idiopathic pulmonary fibrosis (IPF) is a disease characterized by aberrant metabolism, including enhanced glutaminolysis, the conversion of glutamine (Gln) to glutamate by the enzyme glutaminase, GLS-1. IPF fibroblasts acquire an apoptosis-resistant phenotype. In this study, we investigated the effects of Gln metabolism on apoptosis susceptibility in primary IPF fibroblasts. Methods: 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 Gln), the medium was changed into fresh full Gln (2 mM) or reduced Gln (0 and 0.1 mM) for 48 h. In some studies, GLS-1 was silenced by siRNA to inhibit glutaminolysis. Cells were then subjected to staurosporine to induce apoptosis, and both protein and mRNA changes were measured. Histone modification changes and related enzymes were examined under the same conditions, the associations between histone modifications and the promoter regions of apoptosis-related genes were examined by ChIP assays. Results: IPF fibroblasts under reduced Gln conditions demonstrated increased apoptosis susceptibility, as evidenced by significantly higher caspse-3 activity. This apoptosis susceptibility was associated with downregulation of the pro-survival genes, XIAP and survivin, at both the mRNA and protein levels. The repressive histone mark H3K27Me3 showed increased levels in reduced Gln conditions, likely due to decreases in the H3K27Me3 demethylase JMJD3 which requires the Gln metabolite α-KG as a cofactor. ChIP assays demonstrated increased association of XIAP and survivin promoter regions with the repressive mark, H3K27Me3. Conclusions: Our data indicate that glutaminolysis may promote apoptosis resistance of IPF fibroblasts by inducing the pro-survival genes, XIAP and survivin. This epigenetic regulation is mediated via α-KG/JMJD3-dependent regulation of H3K27Me3. This study demonstrates a novel mechanism by which altered metabolism can change cell phenotype through epigenetic regulation, supporting new treatment approaches for IPF.