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Glutaminolysis Epigenetically Regulates Antiapoptotic Gene Expression in Idiopathic Pulmonary Fibrosis Fibroblasts.

ABSTRACT: Fibrotic responses involve multiple cellular processes, including epigenetic changes. Epigenetic changes are sensitive to alterations in the tissue microenvironment such as the flux of tricarboxylic acid (TCA) cycle metabolites. TCA metabolites directly regulate epigenetic states, in part by regulating histone modification-related enzymes. Glutaminolysis is a critical metabolic process by which glutamine is converted to glutamate by glutaminase and then to α-ketoglutarate (α-KG), a TCA cycle metabolite. Idiopathic pulmonary fibrosis (IPF) is a disease characterized by aberrant metabolism, including enhanced glutaminolysis. IPF fibroblasts are apoptosis resistant. In this study, we explored the relationship between glutaminolysis and the resistance to apoptosis of IPF fibroblasts. Inhibition of glutaminolysis decreased expression of XIAP and survivin, members of the inhibitor of apoptosis protein (IAP) family. α-KG is a cofactor for JMJD3 histone demethylase, which targets H3K27me3. In the absence of glutamine, JMJD3 activity in fibroblasts is significantly decreased, whereas H3K27me3 levels are increased. Chromatin immunoprecipitation assays confirmed that JMJD3 directly interacts with XIAP and survivin promoter regions in a glutamine-dependent manner. Exogenous α-KG partially restores JMJD3 function and its interaction with the XIAP and survivin promoter regions under glutamine-deficient conditions. Interestingly, α-KG upregulates XIAP, but not survivin, suggesting differential α-KG-dependent and -independent mechanisms by which glutamine regulates these IAPs. Our data demonstrate a novel mechanism of metabolic regulation in which glutaminolysis promotes apoptosis resistance of IPF fibroblasts through epigenetic regulation of XIAP and survivin.

SUBMITTER: Bai L

PROVIDER: S-EPMC6348715 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Idiopathic Pulmonary Fibrosis (IPF) is a debilitating disease characterized by exaggerated extracellular matrix deposition and aggressive lung structural remodeling. Disease pathogenesis is driven by fibroblastic foci formation, consequent on growth factor overexpression and myofibroblast proliferation. We have previously shown that both CTGF overexpression and myofibroblast formation in IPF cell lines are dependent on RhoA signaling. As RhoA-mediated regulation is also involved in cell cycle progression, we hypothesise that this pathway is key to lung fibroblast turnover through modulation of cyclin D1 kinetic expression. METHODS:Cyclin D1 expression was compared in primary IPF patient-derived fibroblasts and equivalent normal control cells. Quantitative real time PCR was employed to examine relative expression levels of cyclin D1 mRNA; protein expression was confirmed by western blotting. Effects of Rho signaling were investigated using transient transfection of constitutively active and dominant negative RhoA constructs as well as pharmacological inhibitors. Cellular proliferation of lung fibroblasts was determined by BrdU incorporation ELISA. To further explore RhoA regulation of cyclin D1 in lung fibroblasts and associated cell cycle progression, an established Rho inhibitor, Simvastatin, was incorporated in our studies. RESULTS:Cyclin D1 expression was upregulated in IPF compared to normal lung fibroblasts under exponential growth conditions (p < 0.05). Serum deprivation inhibited cyclin D1 expression, which was restored following treatment with fibrogenic growth factors (TGF-beta1 and CTGF). RhoA inhibition, using a dominant negative mutant and a pharmacological inhibitor (C3 exotoxin), suppressed levels of cyclin D1 mRNA and protein in IPF fibroblasts, with significant abrogation of cell turnover (p < 0.05). Furthermore, Simvastatin dose-dependently inhibited fibroblast cyclin D1 gene and protein expression, inducing G1 cell cycle arrest. Similar trends were observed in control experiments using normal lung fibroblasts, though exhibited responses were lower in magnitude. CONCLUSION:These findings report for the first time that cyclin D1 expression is deregulated in IPF through a RhoA dependent mechanism that influences lung fibroblast proliferation. This potentially unravels new molecular targets for future anti-IPF strategies; accordingly, Simvastatin inhibition of Rho-mediated cyclin D1 expression in IPF fibroblasts merits further exploitation.

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Glutaminolysis Epigenetically Regulates Antiapoptotic Gene Expression in Idiopathic Pulmonary Fibrosis Fibroblasts.

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