Radiation Induces Metabolic Dysregulation in Pulmonary Fibroblasts

Abstract

Exposure of the lung to ionizing radiation, such as during radiotherapy, can result in pulmonary fibrosis (PF), which has few treatment options. PF is characterized by an accumulation of extracellular matrix proteins that form scar tissue, resulting in dyspnea, disruption of gas exchange, and even death. We and others have shown that metabolic reprogramming is a hallmark of idiopathic pulmonary fibrosis (IPF). IPF lung tissue, and lung fibroblasts treated with TGF-β, exhibit increased aerobic glycolysis with increased expression of lactate dehydrogenase A (LDHA) and excess production of lactate, leading to reduced extracellular pH that activates latent TGF-β. Here, we hypothesized that ionizing radiation would cause aerobic glycolytic metabolic dysregulation in primary human lung fibroblasts.Methods: Primary human lung fibroblasts (HLFs) from two non-fibrotic donors were seeded and subjected to either no treatment, TGF-β treatment (500 pg/mL), or radiation (3, 5, and 7 Gy). Cell lysates were harvested 2 and 5 days after irradiation for RNA and protein, respectively. Gene and protein expression of metabolic markers were determined by RT-PCR and western blot. TBP (RT-PCR) and GAPDH (western blots) were used as loading controls. Cell viability was estimated immediately prior to cell lysate harvest using Presto Blue.Results: Primary non-fibrotic HLFs exposed to irradiation exhibited significant upregulation of Pyruvate Dehydrogenase Kinase (PDK)1 (0.5 -3-fold, p<0.05), LDHA (1.4-fold, p<0.05), and LDHB (2-fold, p<0.05). The transcription factor FOXO1 exhibited a trend toward increased expression. Cell viability was unaffected by increased radiation dose.Conclusions: Radiation increased fibroblast expression of genes involved in aerobic glycolysis (PDK1, LDHA, LDHB), in a similar pattern to that seen in IPF fibroblasts. FOXO1, which regulates PDK1 and other genes in the glycolytic pathway, was not significantly upregulated. Radiation may alter its activity rather than mRNA levels. The metabolic changes are closely associated with creating a profibrotic extracellular environment in IPF by promoting an acidic environment. This phenomenon in fibrotic fibroblasts is similar to observations of the Warburg effect in cancer cells, where aerobic glycolysis occurs despite the presence of oxygen, allowing growth advantages. Our evidence suggests this phenomenon can be driven by radiation in lung fibroblasts and affirm that glycolytic reprogramming may also be a hallmark of radiation-induced fibrosis. Further understanding of the common mechanisms that create this metabolic shift could provide novel therapeutics for fibrosis treatment.