Iron regulatory protein 2 modulates the switch from aerobic glycolysis to oxidative phosphorylation in mouse embryonic fibroblasts Huihui Lia, Yutong Liua, Longcheng Shangb, Jing Caib, Jing Wua, Wei Zhanga, Xiaojiang Pua, Weichen Donga, Tong Qiaob, and Kuanyu Lia,1 aJiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing 210093, People’s Republic of China; and bDepartment of Vascular Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, People’s Republic of China Edited by Nancy C. Andrews, Duke University School of Medicine, Durham, NC, and approved April 9, 2019 (received for review December 1, 2018) The importance of the role of iron regulatory proteins (IRPs) in In our previous study, we found that the Irp1-orIrp2-null mitochondrial iron homeostasis and function has been raised. To mutation in mouse embryonic fibroblasts (MEFs) caused de- understand how an IRP affects mitochondrial function, we used creased expression of frataxin (Fxn) and iron–sulfur cluster globally Irp2-depleted mouse embryonic fibroblasts (MEFs) and scaffold protein IscU, two important components of the Fe–S found that Irp2 ablation significantly induced the expression of biogenesis machinery (11). Deficiency of Fxn or IscU in human both hypoxia-inducible factor subunits, Hif1α and Hif2α. The in- and mouse cells limits mitochondrial function due to the lack of crease of Hif1α up-regulated its targeted genes, enhancing glycol- sufficient Fe–S clusters (12, 13). Furthermore, IRP depletion- ysis, and the increase of Hif2α down-regulated the expression of induced deficiency of Fxn and IscU specifically adversely af- iron–sulfur cluster (Fe–S) biogenesis-related and electron transport fects the activity of the Fe–S-dependent mitochondrial re- chain (ETC)-related genes, weakening mitochondrial respiration. spiratory chain, while the activities of other Fe–S-dependent Inhibition of Hif1α by genetic knockdown or a specific inhibitor enzymes, such as aconitase and xanthine dehydrogenase, are prevented Hif1α-targeted gene expression, leading to decreased enhanced (11). Strangely, ATP is more highly produced in α Irp2−/− aerobic glycolysis. Inhibition of Hif2 by genetic knockdown or MEFs than in WT (the present study). This result CELL BIOLOGY selective disruption of the heterodimerization of Hif2α and Hif1β seeming paradoxical to the low activity of the electron transport restored the mitochondrial ETC and coupled oxidative phosphory- chain (ETC) and high content of ATP, suggesting a shift of the lation (OXPHOS) by enhancing Fe–S biogenesis and increasing ETC- metabolic pathway in Irp2 ablation cells. related gene expression. Our results indicate that Irp2 modulates Oxidative phosphorylation (OXPHOS) and glycolysis are two the metabolic switch from aerobic glycolysis to OXPHOS that is key metabolic pathways for energy production. The switch from mediated by Hif1α and Hif2α in MEFs. one pathway to another is controlled by a number of factors, including two important transcription factors, HIF1 and HIF2. iron regulatory protein 2 | mitochondrial function | energy metabolism | HIFs are heterogeneous dimers that are mainly composed of hypoxia inducible factors an O2-labile alpha subunit (HIF1α or HIF2α)andastable beta subunit (HIF1β, also known as ARNT). The direct con- α ron is essential for growth and proliferation of mammalian nection between Irp and Hif demonstrates that Hif2 is Icells due to its important roles in protein cofactors, hemes and iron–sulfur clusters (Fe–S), which are involved in a number of Significance biochemical pathways, including hemoglobin synthesis and the mitochondrial respiration chain. Cellular iron homeostasis is Iron regulatory proteins (IRPs) control cellular iron homeostasis. secured by two orthologous iron regulatory proteins (IRPs), Irp2 knockout mice show symptoms of neurological disorders, IRP1 and IRP2, both of which are iron-regulated RNA-binding which are considered to result from impaired mitochondrial proteins that posttranscriptionally control the expression of a activity. To explore the involvement of Irp2 in mitochondrial series of iron-related genes, such as ferritin, transferrin receptor function, we examined the metabolic pathways of Irp2- 1(TfR1), ferroportin 1 (FPN1), DMT1, and eALAS (1, 2). When depleted mouse embryonic fibroblasts. We found that Irp2 cells are iron-deficient in the labile pool, IRPs bind iron- deficiency switches cellular metabolic pathways from oxidative responsive elements (IREs) located in the 5′-UTR of ferritin phosphorylation (OXPHOS) to aerobic glycolysis. We further and FPN1 mRNA to inhibit its translation, which reduces iron revealed that Irp2 deficiency induces the expression of Hif1α storage and export, and the IRE in the 3′-UTR of TfR1 and and Hif2α; Hif1α enhances aerobic glycolysis by upregulating DMT1, which stabilizes the mRNA to facilitate iron import. its target genes related to the glycolytic pathway, and Hif2α When cells are iron-abundant, IRP1 is converted to a [4Fe-4S]- suppresses mitochondrial Fe–S biosynthesis and OXPHOS. This containing aconitase, and IRP2 is removed through iron- identified mechanism implies that high-energy-need tissues, mediated proteasomal degradation (3, 4), which increases ferri- such as the central nervous system, could be affected when tin and FPN1 translation and promotes TfR1 and DMT1 mRNA Irp2 is deficient, leading to neurological disorders. degradation, preventing additional iron absorption and avoiding excess iron-induced injury. Author contributions: H.L. and K.L. designed research; H.L., Y.L., L.S., J.C., J.W., W.Z., X.P., Studies have shown that mice lacking Irp2 have abnormal iron and W.D. performed research; T.Q. and K.L. contributed new reagents/analytic tools; H.L., contents in several tissues and develop microcytic anemia and T.Q., and K.L. analyzed data; and H.L. and K.L. wrote the paper. erythropoietic protoporphyria (5, 6). Irp2 knockout mice also The authors declare no conflict of interest. have symptoms of neurological disorders (7–9) due to the This article is a PNAS Direct Submission. functional iron starvation in brain and spinal cord. This func- Published under the PNAS license. tional iron starvation is therefore considered to be causative of 1To whom correspondence should be addressed. Email: [email protected]. the impaired mitochondrial activity (10). However, the exact This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mechanism by which Irp2 sustains normal mitochondrial func- 1073/pnas.1820051116/-/DCSupplemental. tion is still unclear. Published online April 30, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1820051116 PNAS | May 14, 2019 | vol. 116 | no. 20 | 9871–9876 Downloaded by guest on September 28, 2021 posttranscriptionally regulated by Irp1 through binding the IRE in the 5′-UTR of Hif2α mRNA (14, 15). Irp1 ablation mice de- velop polycythemia, cardiac fibrosis, and pulmonary hyperten- sion, which are attributed to a high level of Hif2α, which mediates the up-regulation of erythropoietin (16–18). Although Hif2α is up-regulated in Irp2-depleted cells (18), the physiolog- ical roles of both Hifs in Irp2 ablation mice remain unknown. Here, we address the role of up-regulated Hif1α and Hif2α in −/− Irp2 MEFs in regard to energy metabolism. Using MEFs in which Irp2 is globally depleted, we demonstrated that increased Hif1α enhanced glycolysis by targeting a number of glycolytic pathway-related genes, while increased Hif2α inhibited mito- chondrial OXPHOS by decreasing, likely indirectly, the expres- sion of Fxn and IscU and affecting the mitochondrial Fe–S cluster assembly and by decreasing the expression of ETC sub- units and weakening OXPHOS. Therefore, Irp2 deficiency switches energy metabolism from OXPHOS to glycolysis. Results Irp2 Ablation-Induced Mitochondrial Dysfunction Is Associated with the Metabolic Switch from OXPHOS to Aerobic Glycolysis in MEFs. IRPs have been demonstrated to be important for mitochondrial iron supply and function (19). Consistent with this finding, we and other groups revealed in vivo and in vitro that a deficit of available iron and reduction of mitochondrial Fe–S biogenesis can be key factors in mitochondrial dysfunction in general (20) and in Irp2 ablated cells (10, 11). Here, we confirmed that the activities of mitochondrial complexes I, II, and III significantly −/− decreased in Irp2 MEFs compared with WT (Fig. 1A), which is consistent with the levels of the Fe–S-containing subunits Ndufs1 (of complex I), SdhB (of complex II), and Uqcrfs1 (of Fig. 1. Irp2 ablation-induced mitochondrial dysfunction is associated with −/− complex III) in Irp2 cells (Fig. 1B and SI Appendix, Fig. S1). In enhanced aerobic glycolysis in MEFs. (A) Activities of ETC complexes in Irp2- deficient MEFs. CI, CII, and CIII, complexes I, II, and III. (B) Western blot addition, the amounts of other mitochondrial proteins, such as analysis of mitochondrial proteins, including Ndufs1 (a subunit of CI), SdhB (a cytochrome C (CytC, an intermembrane protein) and ferroche- −/− subunit of CII), Uqcrfs1 (a subunit of CIII), Fech (a matrix enzyme ferroche- latase (Fech, a matrix protein), also decreased in Irp2 cells latase), CytC (an intermembrane space protein cytochrome C), and Cs (a (Fig. 1B and SI Appendix, Fig. S1). To further detect any broad matrix non-Fe–S citrate synthase). A representative image set is presented. effect of Irp2 deficiency on mitochondrial function, we measured Actin was used