Cellular IAP (cIAP) Family Proteins Regulate TGF-β1 Induced Metabolic Reprogramming of Lung Fibroblasts

Abstract

The inappropriate survival and persistent activation of myofibroblasts impedes the normal resolution of wound repair and promotes fibrosis. Myofibroblasts from patients with fibrotic lung disease have increased expression of Inhibitor of Apoptosis family proteins (IAPs), and pro-fibrotic stimuli, including Transforming Growth Factor Beta-1 (TGF-β1) induce IAP family protein expression. Pharmacologic inhibition of the "cellular" IAPs (cIAP-1, cIAP-2 and XIAP) diminishes lung fibrosis in murine models, and knockdown of X-linked IAP (XIAP) sensitizes fibroblasts to undergo apoptosis. TGF-β1 also induces "metabolic reprogramming", with aerobic glycolysis in normal fibroblasts. Moreover, glutaminase-dependent alpha-ketoglutarate production maintains XIAP expression in fibroblasts while glutaminase deficiency leads to decreased XIAP and enhanced apoptosis. The mechanisms that underlie aerobic glycolysis in fibroblasts, and how metabolic reprogramming affects fibroblast phenotypes, have not been determined. We hypothesized that cellular IAPs, and XIAP specifically, would regulate the metabolic reprogramming of lung fibroblasts by TGF-β1. Methods: Normal human lung fibroblasts (IMR-90) were cultured to 60% confluence and growth arrested for 24 hours prior to treatment with TGF-β1 (2 ng/ml) for 24-48 hours. As indicated, cells were transfected with siRNA targeting individual IAPs. IAP expression was assessed by quantitative real-time rtPCR. Mitochondrial Respiration and glycolysis were assessed using Seahorse xfe96 analyzer. Results: cIAP-1 and XIAP increased rapidly following TGF-β1 treatment and the increased levels persisted for 24-48 hours. In contrast, cIAP-2 expression decreased at 24 and 48 hours. Using siRNA targeting each of the cellular IAPs, we examined their role in mitochondrial respiration and glycolysis following TGF-β1 exposure. As expected, TGF-β1 increased respiration and ATP production in parallel with increased glycolysis. Silencing XIAP or cIAP-2 enhanced TGF-β1-stimulated mitochondrial respiration. In contrast, silencing cIAP-1 diminished basal and TGF-β1-stimulated respiration. Silencing cIAP-1 also prevented the increased glycolysis following TGF-β1. However, silencing of XIAP or cIAP-2 had no effect on glycolysis. Conclusions: Metabolic reprogramming has emerged as a characteristic phenotype of TGF-β1-activated myofibroblasts, and our studies demonstrate a novel role for cellular IAP family proteins in this process. Specifically, we identify a critical role for cIAP-1 in TGF-β1-induced aerobic glycolysis. Surprisingly, these studies also suggest that XIAP and cIAP-2 suppress TGF-β1-stimulated mitochondrial respiration and have no role in TGF-β1 stimulated glycolysis. Taken together, these findings indicate complex counter-regulatory roles for the structurally homologous cellular IAP family members in TGF-β1-stimulated aerobic glycolysis in lung fibroblasts.