glutaminase and Pulmonary-Fibrosis

Fibrosis is an energy-intensive process requiring the activation of fibroblasts to myofibroblasts, resulting in the increased synthesis of extracellular matrix proteins. Little is known about the transcriptional control of energy metabolism in cardiac fibroblast activation, but glutaminolysis has been implicated in liver and lung fibrosis. Here we explored how pro-fibrotic TGFβ and its effector scleraxis, which drive cardiac fibroblast activation, regulate genes involved in glutaminolysis, particularly the rate-limiting enzyme glutaminase (GLS1). The GLS1 inhibitor CB-839 attenuated TGFβ-induced fibroblast activation. Cardiac fibroblast activation to myofibroblasts by scleraxis overexpression increased glutaminolysis gene expression, including GLS1, while cardiac fibroblasts from scleraxis-null mice showed reduced expression. TGFβ induced GLS1 expression and increased intracellular glutamine and glutamate levels, indicative of increased glutaminolysis, but in scleraxis knockout cells, these measures were attenuated, and the response to TGFβ was lost. The knockdown of scleraxis in activated cardiac fibroblasts reduced GLS1 expression by 75%. Scleraxis transactivated the human GLS1 promoter in luciferase reporter assays, and this effect was dependent on a key scleraxis-binding E-box motif. These results implicate scleraxis-mediated GLS1 expression as a key regulator of glutaminolysis in cardiac fibroblast activation, and blocking scleraxis in this process may provide a means of starving fibroblasts of the energy required for fibrosis.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Fibroblasts; Glutaminase; Mice; Myofibroblasts; Pulmonary Fibrosis; Transforming Growth Factor beta

In the current work we show that the profibrotic actions of TGF-β are mediated, at least in part, through a metabolic maladaptation in glutamine metabolism and how the inhibition of glutaminase 1 (GLS1) reverses pulmonary fibrosis. GLS1 was found to be highly expressed in fibrotic vs normal lung fibroblasts and the expression of profibrotic targets, cell migration, and soft agar colony formation stimulated by TGF-β required GLS1 activity. Moreover, knockdown of SMAD2 or SMAD3 as well as inhibition of PI3K, mTORC2, and PDGFR abrogated the induction of GLS1 by TGF-β. We further demonstrated that the NAD-dependent protein deacetylase, SIRT7, and the FOXO4 transcription factor acted as endogenous brakes for GLS1 expression, which are inhibited by TGF-β. Lastly, administration of the GLS1 inhibitor CB-839 attenuated bleomycin-induced pulmonary fibrosis. Our study points to an exciting and unexplored connection between epigenetic and transcriptional processes that regulate glutamine metabolism and fibrotic development in a TGF-β-dependent manner.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Cell Movement; Cells, Cultured; Female; Fibroblasts; Gene Expression Regulation; Glutaminase; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Signal Transduction; Sirtuins; Smad Proteins; Transforming Growth Factor beta

It has been increasingly recognized lately that aberrant cellular metabolism plays an important role in the pathogenesis of pulmonary fibrosis. In our previous systemic studies, we found that human lung myofibroblasts undergo glutaminolytic reprogramming, which is mediated by an increased expression of glutaminase (Gls) 1. We showed that augmented glutaminolysis critically regulates collagen production by promoting its stabilization in human lung myofibroblasts. Our study indicates that lung fibroblast Gls1 is a promising therapeutic target for this disease. In this investigation, we primarily focused on delineating the

Topics: Animals; Benzeneacetamides; Bleomycin; Cell Line; Collagen; Enzyme Induction; Fibroblasts; Glutaminase; Lung; Male; Mice; Mice, Inbred C57BL; Molecular Targeted Therapy; Pulmonary Fibrosis; RNA Interference; RNA, Messenger; RNA, Small Interfering; Thiadiazoles; Transforming Growth Factor beta1

Glutaminolysis is the metabolic process of glutamine, aberration of which has been implicated in several pathogeneses. Although we and others recently found a diversity of metabolic dysregulation in organ fibrosis, it is unknown if glutaminolysis regulates the profibrotic activities of myofibroblasts, the primary effector in this pathology. In this study, we found that lung myofibroblasts demonstrated significantly augmented glutaminolysis that was mediated by elevated glutaminase 1 (Gls1). Inhibition of glutaminolysis by specific Gls1 inhibitors CB-839 and BPTES as well as Gls1 siRNA blunted the expression of collagens but not that of fibronectin, elastin, or myofibroblastic marker smooth muscle actin-α. We found that glutaminolysis enhanced collagen translation and stability, which were mediated by glutaminolysis-dependent mTOR complex 1 activation and collagen proline hydroxylation, respectively. Furthermore, we found that the amount of the glutaminolytic end product α-ketoglutarate (α-KG) was increased in myofibroblasts. Similar to glutaminolysis, α-KG activated mTOR complex 1 and promoted the expression of collagens but not of fibronectin, elastin, or smooth muscle actin-α. α-KG also remarkably inhibited collagen degradation in fibroblasts. Taken together, our studies identified a previously unrecognized mechanism by which a major metabolic program regulates the exuberant production of collagens in myofibroblasts and suggest that glutaminolysis is a novel therapeutic target for treating organ fibrosis, including idiopathic pulmonary fibrosis.

Topics: Actins; Animals; Benzeneacetamides; Cells, Cultured; Collagen; Disease Models, Animal; Elastin; Enzyme Activation; Fibronectins; Glutaminase; Glutamine; Humans; Hydroxylation; Ketoglutaric Acids; Mice; Mice, Inbred C57BL; Myofibroblasts; Proline; Pulmonary Fibrosis; RNA Interference; RNA, Small Interfering; Sulfides; Thiadiazoles; TOR Serine-Threonine Kinases