Iron Regulatory Protein 2 Modulates the Switch from Aerobic Glycolysis to Oxidative Phosphorylation in Mouse Embryonic Fibroblasts

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.

www.pnas.org/cgi/doi/10.1073/pnas.1820051116 PNAS Latest Articles | 1of6 Downloaded by guest on September 25, 2021 posttranscriptionally regulated by Irp1 through binding the IRE in the 5′-UTR of Hif2α mRNA (14, 15). Irp1 ablation mice develop polycythemia, cardiac fibrosis, and pulmonary hypertension, 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 mitochondrial OXPHOS by decreasing, likely indirectly, the expression of Fxn and IscU and affecting the mitochondrial Fe–S cluster assembly and by decreasing the expression of ETC subunits 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 as a loading control. (C) Sensitivity of Irp2−/− cells to ETC −/− the sensitivity of Irp2 cells to various inhibitors of mitochon- complex inhibitors, including rotenone (10 μM, inhibitor of complex I), −/− drial complexes. The results showed that the viability of Irp2 cells oxaloacetic acid (OAA) (100 μM, inhibitor of complex II), and antimycin A μ significantly decreased compared with WT (Fig. 1C), suggesting (10 M, inhibitor of complex III). (D) MMP detected using JC-10. Green that these cells are more sensitive to perturbations of mito- fluorescence represents JC-10 monomers, and red fluorescence represents JC-10 aggregates. The ratio of red fluorescence to green fluorescence rep- chondrial function after Irp2 deprivation. The mitochondrial resents the level of the mitochondrial membrane potential. (E) Growth membrane potential (MMP), as measured using a specific and −/− −/− −/− curves of WT and Irp2 cells. (F) Intracellular ATP content of WT and Irp2 sensitive dye, JC-10, was much lower in Irp2 cells than in cells in the growth phase (day 2 after subculture). (G) A representative set −/− WT cells (Fig. 1D), suggesting that mitochondria in Irp2 cells for proteins Hk2, Glut1, LdhA, LdhB, Pdh(-E1α), and p-Pdh(-E1α (pSer232)) are more depolarized. revealed by Western blot analysis. (H) Levels of medium lactic acid in WT and − − Interestingly, although Irp2 deletion seriously weakened mi- Irp2 / cells cultured in medium containing 4.5 g/L glucose (H, high) or 1.0 g/L −/− tochondrial function, the growth of Irp2 cells was not signifi- glucose (L, low). (I) A representative set of Western blot analyses of glycolytic cantly retarded, and the difference between WT and mutant was pathway-related proteins (Hk2, Glut1, LdhA, and LdhB) and oxidative only pronounced on the fourth day due to insufficient nutrients phosphorylation pathway-related proteins (Pdh, Ndufs1, and Uqcrfs1). Actin was used as a loading control. Representative blots from n = 3 experiments (Fig. 1E). Surprisingly, the level of ATP significantly increased in ± Irp2−/− are shown (each with duplicates). Values represent the mean SEM. One- cells compared with WT in the growth phase (day 2) (Fig. way ANOVA (H) or Student’s t test (A, C, E, and F) was performed. *P < 0.05, 1F). We then speculated that aerobic glycolysis was enhanced to **P < 0.01, ***P < 0.001, mutant vs. WT. ##P < 0.01, low vs. high concen- provide enough ATP for cell growth. Therefore, we detected the tration of glucose in medium. levels of several proteins involved in glycolysis, such as hexoki- nase 2 (HK2), glucose transporter 1 (Glut1), and lactate dehydrogenase A/B (LdhA/B). The expression of these proteins results, we cultured cells in medium containing a high (4.5 g/L) −/− was significantly increased in Irp2 cells (Fig. 1G and SI Ap- or low (1.0 g/L) concentration of glucose for 3 d and measured pendix the content of lactic acid, a by-product of the postglycolysis , Fig. S1). We also detected pyruvate dehydrogenase −/− (Pdh), a vital regulatory enzyme that catalyzes the conversion of pathway. As shown in Fig. 1H, Irp2 cells always produced and pyruvate into acetyl-CoA and connects glycolysis to the TCA secreted more lactic acid than WT cells under both glucose cycle. The protein level of Pdh was significantly reduced in concentration conditions and in a concentration-dependent −/− Irp2 cells (Fig. 1G and SI Appendix, Fig. S1). However, manner. The protein levels of Hk2, Glut1, and LdhA/B all −/− phosphorylation of the E1α subunit of Pdh (p-Pdh–E1α consistently increased in Irp2 cells under a high glucose con- (pSer232)), which leads to inactivation of the Pdh complex en- centration (Fig. 1I). We then evaluated cellular OXPHOS by zymatic activity, was enhanced (Fig. 1G and SI Appendix, Fig. detecting the levels of related proteins. The expression of Pdh, S1), suggesting that glycolytic metabolism was favored over Ndufs1, and Uqcrfs1 was not affected by different concentrations −/− mitochondria-dependent metabolism. To further verify these of glucose in WT or Irp2 cells, although these proteins all

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1820051116 Li et al. Downloaded by guest on September 25, 2021 −/− expressed less in Irp2 cells (Fig. 1I). These results strongly and a lower maximal mitochondrial capacity than WT cells after suggest that Irp2 deficiency promotes cellular aerobic glycolysis treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone −/− and suppresses OXPHOS. (FCCP), suggesting that Irp2 cells are less oxidative, consistent To verify this hypothesis, the oxygen consumption rate (OCR) with the lower mitochondria-derived ATP content. Simulta- −/− and extracellular acidification rate (ECAR), as indicators of neously, Irp2 cells had a much higher ECAR after glucose and mitochondrial respiration and the glycolytic rate, respectively, oligomycin treatment (Fig. 2 C and D), suggesting that they are were measured using an Agilent Seahorse Analyzer. As illustrated more glycolytic. Collectively, these results demonstrated that −/− in Fig. 2 A and B, Irp2 cellshadlowerrestingOCRorOXPHOS Irp2 deficiency induced a metabolic switch from OXPHOS to aerobic glycolysis. To further confirm that the metabolic switch was due to Irp2 deficiency, human IRP1 or IRP2, homologs of mouse Irp1 and Irp2, respectively, was expressed in Irp2-depleted MEFs to assess the recovery of iron and energy metabolism. Cellular iron metabolism was evaluated (SI Appendix, Fig. S2), and the results supported the conserved iron regulatory function of human IRP1 and IRP2. Evaluation of energy metabolism revealed that the expression of IRP2 increased the activities of respiration com- −/− plexes I, II, and III, while the expression of IRP1 in Irp2 cells only increased the activities of complexes I and II (Fig. 2E). This −/− result was in agreement with the sensitivity of Irp2 cells to inhibitors of mitochondrial ETC complexes (Fig. 2F). We also found that only IRP2, and not IRP1, significantly improved the −/− MMP of Irp2 cells (Fig. 2G). Biochemical evidence revealed that all of the tested OXPHOS-related protein levels, such as −/− Pdh, Ndufs1, and Uqcrfs1, significantly increased in Irp2 cells after IRP2 expression, but only the level of Pdh increased after IRP1 expression (Fig. 2H and SI Appendix, Fig. S3). In- terestingly, the levels of the postglycolysis-related proteins LdhA CELL BIOLOGY and LdhB remained high after either IRP1 or IRP2 expression (Fig. 2H and SI Appendix, Fig. S3). However, the contents of −/− lactic acid in both the cell lysate and medium of Irp2 cells were reduced by IRP2 expression, not by IRP1 expression in medium (Fig. 2I). Thus far, we have provided evidence that Irp2 can shift cellular respiration in favor of OXPHOS over aerobic glycolysis.

Irp2 Absence-Induced Up-Regulation of Hif2α in MEFs Affects Mitochondrial Biogenesis. We further investigated the mechanism that drives the switch from OXPHOS to glycolysis in Irp2- deprived MEFs. HIF is a key transcriptional factor in regulating a number of genes involved in glycolytic respiration, including some of the genes tested in this study. Our previous study (11) and this work revealed that Irp2 deficiency resulted in a signif- icant reduction of the iron content in MEFs (SI Appendix, Fig. −/− S2), which might stabilize Hif1α and Hif2α in Irp2 cells. In- deed, the results shown in Fig. 3A and the SI Appendix, Fig. S4 A −/− and B, confirmed our hypothesis. We then treated Irp2 cells with specific inhibitors of Hif1α (PX-478) and Hif2α (PT-2385). PX-478 transcriptionally and translationally inhibits Hif1α expression, and PT-2385 selectively disrupts the heterodimerization of Hif2α with Hif1β, although their mechanisms of action have yet to be fully elucidated (see review in ref. 21). The con- Fig. 2. Human IRP2 rescues Irp2 ablation-induced mitochondrial dysfunction centrations of the drugs were optimized (SI Appendix, Fig. S4C), and reverses energy metabolism in MEFs. (A) Profiles of the OCR in WT and −/− μ μ and the effects of the inhibition were confirmed by the down- Irp2 cells. Oligomycin, 1 M; FCCP, 1 M; rotenone/antimycin (Rot/AA), regulation of the Hif-targeted genes LdhA, endothelin 1 (Edn1), 0.5 μM. (B) The calculated OCR for basal and maximal respiration and ATP −/− and Glut1 (Fig. 3B). The results suggested that LdhA and Glut1 production. (C) Profiles of the ECAR in WT and Irp2 cells.. Glucose, 10 mM; Edn1 oligomycin, 1 μM; 2-deoxyglucose (2-DG), 50 mM. (D) The calculated ECAR for were mainly targeted by Hif1 and was mainly targeted by glycolysis and the glycolytic capacity. (E) Enzymatic activities of CI, CII, and CIII Hif2 in MEFs. Surprisingly, Fxn and IscU expression was sig- determined in Irp2-deficient MEFs after transfection with pcMV-HA-IRP1 or nificantly up-regulated by PT-2385 but not by PX-478 (SI Ap- pDEST-his-IRP2. (F) Sensitivities to ETC complex inhibitors after IRP1 or IRP2 pendix, Fig. S4C). We further examined a number of genes − − expression in Irp2 / cells. The treatment with complex inhibitors was the same involved in iron metabolism and OXPHOS. Inhibition of Hif1α −/− −/− as in Fig. 1. (G) MMP of Irp2 cells after expression of IRP1 or IRP2. *P = 0.0443; in Irp2 cells did not change the protein levels of iron-related # = = P 0.0274; NS, P 0.0819. (H) Protein levels of IRP1, IRP2, LdhA, LdhB, Pdh, genes, such as Fxn and IscU, or mitochondrion-related genes, Ndufs1, and Uqcrfs1 determined by Western blot analysis. (I)Levelsoflacticacid −/− such as Pdh, Ndufs1, SdhB, and Uqcrfs1 (Fig. 3C and SI Ap- inthemediumorcelllysateofIrp2 cells after expression of IRP1 or IRP2. pendix α Irp2−/− Actin was used as a loading control in Western blot analysis. Values represent , Fig. S5). By contrast, suppression of Hif2 in cells the mean ± SEM (n = 3–5, each with duplicates). In B, D, E, F, G,andI,*P < 0.05, significantly increased the protein levels of Pdh, Ndufs1, SdhB, **P < 0.01, ***P < 0.001, mutant vs. WT; #P < 0.05, ##P < 0.01, ###P < 0.001, IRP Uqcrfs1, Fxn, and IscU (Fig. 3D and SI Appendix, Fig. S6). The rescue vs. nonrescue. NS, no significance, IRP1 rescue vs. nonrescue. effects of the specific inhibitors PX-478 and PT-2385 were also

Li et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 25, 2021 To reveal the biochemical basis of the above observed phenotypes, we measured the activities of mitochondrial ETC complexes I, II, and III and found that they were all significantly increased by inhibition of Hif2α alone or by inhibition of both −/− Hif1α and Hif2α in Irp2 cells but not by inhibition of Hif1α alone (Fig. 4E). Although the TfR1 level remained constant for iron import, ferritin expression was significantly increased (Fig. 4F), in line with the cytoplasmic labile iron pool (LIP) level (SI Appendix,Fig.S8).

Fig. 3. Irp2 deficiency-induced mitochondrial dysfunction is mediated by up- regulated Hif2α.(A) Irp2 deficiency-induced expression of Hif1α and Hif2α determined by qRT-PCR (Left) and Western blot analysis (Right). (B) Effects of Hif1α and Hif2α inhibition by PX-478 (a specific inhibitor of Hif1α,50μM) and PT-2385 (a specific inhibitor of Hif2α,50μM), respectively, on their target genes LdhA, endothelin 1 (Edn1), and Glut1 in Irp2−/− cells. (C and D)Expressionof OXPHOS or Fe–S biogenesis-related proteins, Pdh, Ndufs1, SdhB, Uqcrfs1, IscU, and Fxn, after inhibition of Hif1α by PX-478 (50 μM) (C) and inhibition of Hif2α by PT-2385 (10–50 μM) (D) determined by Western blot analysis. DMSO as a vehicle of PT-2385 was added at an identical volume when cells were treated. A representative image set is presented, and the quantitative data of the protein levels are shown in the SI Appendix, Figs. S5 and S6. Values represent the mean ± SEM (n = 3, each with duplicates). *P < 0.05, **P < 0.01, ***P < 0.001, mutant vs. WT. #P < 0.05, ##P < 0.01, with inhibitor vs. without inhibitor. NS, no significance, with inhibitor vs. without inhibitor.

verified by an unspecific inhibitor, 2-methoxyestradiol (SI Ap- pendix, Fig. S7), suggesting that Irp2 absence-induced up- regulation of Hif2α in MEFs inhibits mitochondria-dependent metabolism.

Both Hif1 and Hif2 Collaboratively Mediate the Metabolic Switch of Irp2−/− Cells. To further demonstrate whether the increased expression of Hif1α and Hif2α induced by Irp2 deficiency was the cause of the cellular metabolic shift, we examined whether the Fig. 4. Both Hif1α and Hif2α collaboratively mediate the metabolic switch of phenotypes could be reversed after inhibition of Hif1α and −/− −/− −/− Irp2 cells. (A) Profiles of the OCR, reflecting OXPHOS activity in Irp2 cells Hif2α. We measured the OCR and ECAR in Irp2 cells after after treatment with PX-478 (50 μM) or PT-2385 (50 μM) for 24 h. (B)Thecal- treatment with PX-478 or PT-2385. The results showed that culated OCR for basal and maximal respiration and ATP production. (C) Profiles − − basal respiration, maximal respiration, and OXPHOS-dependent of the ECAR, reflecting glycolytic activity in Irp2 / cells after treatment with PX- α 478 or PT-2385 for 24 h. (D) The calculated ECAR for glycolysis and glycolytic ATP production were efficiently reversed by inhibiting Hif2 but −/− α A B capacity. (E) Activities of ETC complexes CI, CII, and CIII in Irp2 MEFs after not by inhibiting Hif1 (Fig. 4 and ). By contrast, both gly- treatment with PX-478, PT-2385, or both PX-478 and PT-2385 for 24 h. (F) colysis and the glycolysis capacity were significantly suppressed Protein levels of a series of genes involved in either aerobic glycolysis or by inhibition of Hif1α but not by inhibition of Hif2α (Fig. 4 C and OXPHOS determined by Western blot analysis after inhibition of Hif1α and/or −/− −/− D). These results further validate that Irp2 deficiency enhances Hif2α in Irp2 cells. (G) Levels of lactic acid in the medium of Irp2 MEFs after treatment with PX-478 and/or PT-2385 for 24 h. (H) Intracellular ATP content of α −− glycolysis by inducing the expression of Hif1 and suppresses Irp2 / cells after treatment with PX-478 and/or PT-2385. Values represent the OXPHOS and mitochondrial biogenesis by inducing the expression mean ± SEM (n = 3–5, each with duplicates). *P < 0.05, **P < 0.01, ***P < 0.001, of Hif2α, thereby switching energy metabolism in MEFs. mutant vs. WT; #P < 0.05, ##P < 0.01, ###P < 0.001, with inhibitor vs. without inhibitor.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1820051116 Li et al. Downloaded by guest on September 25, 2021 In accordance with this result, the expression of Fxn, IscU, target both IRP1 and IRP2 for degradation (4, 26). A strong −/− Ndufs1, SdhB, and Uqcrfs1 significantly increased in Irp2 cells induction of FBXL5 was observed when the cytosolic Fe–S as- after inhibiting Hif2α, and the effects were even more profound sembly system was impaired, which contributed to the degrada- with simultaneous inhibition of Hif1α and Hif2α (Fig. 4F). The tion of the Irp1 protein (26). This degradation likely results in −/− expression of LdhA and HK2 was reduced (Fig. 4F) after Hif2α up-regulation in Irp2 cells, in line with the unaltered treatment with PX-478, which was in agreement with the protein level of Hif2α after the addition of iron (SI Appendix, drastically diminished production of lactic acid in the medium, Fig. S11). These results suggest that Irp1 reduction rather than while no change was observed after treatment with PT-2385 iron starvation is, at least partially, causative of Hif2α stabiliza- −/− alone (Fig. 4G). The combined treatment of PX-478 and PT- tion in Irp2 cells. This result was verified by exogenous ex- −/− 2385 showed an effect on the production of lactic acid similar pression of IRP1 in Irp2 cells, which reversed the Hif2α to that with PX-478 alone (Fig. 4G). Furthermore, the ATP protein levels to some extent (SI Appendix, Fig. S2), very likely content correlated very well with the levels of lactic acid and through IRP1–IRE binding to the 5′-UTR of Hif2α mRNA to increased when Hif2 was inhibited (Fig. 4H). These results inhibit translation. −/− further prove that the effects of Hif1 and Hif2 are independent Inhibition of Hif1α in Irp2 cells suppressed the Hif1α target and that aerobic glycolysis is a major metabolic pathway in genes HK2, Glut1,andLdhA. As a result, aerobic glycolysis −/− Irp2 MEFs. was repressed, and the lactic acid levels decreased, whereas The effects of Hif1α and Hif2α on the metabolic switch were OXPHOS-related genes and enzyme activities did not respond to further validated by a shRNA or siRNA knockdown approach. Hif1α inhibition. Thus, the decreased ATP content caused by The knockdown efficiency was first evaluated (SI Appendix, Fig. Hif1α inhibition is glycolysis-dependent. By contrast, Hif2α in- −/− S9 A and B), and the best shRNA and siRNA were used to knock hibition in Irp2 cells drastically up-regulated the OXPHOS- down Hif1α and Hif2α, respectively. The Western blot results related genes Ndufs1, SdhB, and Uqcrfs1, leading to the resto- showed that the expression of the Hif1-targeted genes HK2 and ration of the activities of complexes I–III. Meanwhile, the ATP LdhA was significantly reduced after Hif1α was knocked down content increased further, proving that Irp2 depletion-induced with siRNA (SI Appendix, Fig. S9C). The consequence was the Hif2α represses OXPHOS and reduces mitochondrion-dependent reduced production of lactic acid (SI Appendix, Fig. S9D). Sim- ATP production. This effect is likely attributed to the suppres- ilarly, the expression of Hif2-targeted genes was reduced when sion of IscU and Fxn (this study and refs. 11 and 27). IscU has Hif2α was knocked down with shRNA (SI Appendix, Fig. S9E). been revealed to be a member of the miR-210 regulon during

In line with the drug inhibition of Hif2, Fe–S biogenesis-related hypoxia and adversely controls mitochondrial metabolism (28). CELL BIOLOGY (Fxn and IscU) and OXPHOS-related (complex I subunit The promoter of miR-210 contains a hypoxic responsive element Ndufs1, complex II subunit SdhB, and complex III subunit (HRE) for Hif binding (29). Therefore, down-regulation of IscU −/− Uqcrfs1) genes were up-regulated (SI Appendix, Fig. S9F). As in Irp2 MEFs is presumably through the miR-210–Hif axis. anticipated, the ATP content increased further (SI Appendix, Hif1 and Hif2, both in combination (30) and individually (31), Fig. S9G). Combining the results from the drug inhibition and have been verified to target miR-210 in various tumor cells. −/− genetic approaches, we concluded that the Irp2 ablation- Remarkably, in Irp2 MEFs, only the inhibition of Hif2α, not induced metabolic switch is mediated by up-regulated Hif1α that of Hif1α, increased IscU expression, suggesting that miR- and Hif2α. 210 is regulated by Hif2 in MEFs. Strikingly, Fxn exhibits very similar responses to Irp2 depletion (ref. 11 and this study), Hif Discussion inhibition (this study), and iron regulation (12, 32) as IscU. In this study, we first found that Irp2-deficient MEFs had nearly Human FXN has been reported to be directly regulated by Hif1, normal growth despite their significantly low ETC activity. In- not by Hif2 (33). Mouse Fxn, in contrast, is controlled by Hif2, terestingly, the ATP content was relatively higher in mutant cells. not by Hif1 (34). The up-regulation of FXN by both Hifs is −/− We further discovered that Irp2 cells favored aerobic glycol- thought to occur through the binding of HIF-HRE to the promoter ysis over OXPHOS, which was triggered by up-regulated Hif1α region of FXN. However, we found that Hif2, but not Hif1, down- and Hif2α. Inhibition of both Hifs suppressed aerobic glycolysis regulated the expression of Fxn in MEFs. The mechanism needs and enhanced OXPHOS, thereby switching respiration from to be investigated further. −/− aerobic glycolysis to OXPHOS in Irp2 cells (illustrated in SI Consistently, we found here and previously (11) that Irp1 and Appendix, Fig. S10). This illustration is in line with previous Irp2 had distinct impacts on mitochondrial metabolism, although studies, in which Hif1 and Hif2, while sharing structural simi- they are interchangeable in terms of iron metabolism. The rescue larity and common target genes, have unique targets involved in experiments (Fig. 2E) showed that either IRP1 or IRP2 could different pathways (22, 23). reverse the Irp2 deficiency-induced enzymatic defects of com- Under normal oxygen conditions, prolyl hydroxylase domain plexes I and II, whereas the activity of complex III could only be enzymes (PHDs), which are master regulators of the hypoxia reversed by IRP2 expression. Comparable results have been response (24), hydroxylate HIFα subunits at conserved prolines, reported in which tempol treatment restored complex I activity −/− leading to HIFα degradation by the proteasome. PHD activity of Irp2 mice by converting Irp1 from the cytosolic aconitase to requires iron binding at an active site. Therefore, Hif1α is sta- the IRE binding form for iron uptake to improve the neurode- −/− bilized by iron starvation, which is induced by Irp2 ablation in this generative symptoms of Irp2 mice (35). Tempol-induced iron study. Hif1α contains a unique transactivation domain that al- bioavailability, presumably, also reduces Hif1α stability. The lows preferential activation of hypoxia-responsive glycolytic important role of Hif1α in neurodegenerative diseases has been genes; its downstream genes, such as HK2, Glut1, and LdhA, are proposed, where Hif1α can be considered to be a therapeutic −/− up-regulated in Irp2 cells. This result was proven by the ad- target (36). Moreover, inhibition of Hif1α blocked glycolysis, dition of iron, which reduced Hif1α protein levels (SI Appendix, which is the main metabolic pathway to generate ATP and lactic −/− Fig. S11). However, up-regulated Hif2α did not respond to iron acid in Irp2 cells. The high level of lactic acid or methyl- treatment (SI Appendix, Figs. S11A and S12B). Hif2α stabiliza- glyoxal, a highly reactive dicarbonyl compound inevitably formed tion seems more complex because it can be regulated both by as a by-product of glycolysis, might be toxic to neuronal function iron, similar to Hif1α, and directly by Irps (15, 25), probably (37, 38). Despite the astrocyte-neuron lactate shuttle hypothesis mainly by Irp1 (14). Interestingly, the Irp1 levels were reduced in (39) and the high expression of lactate dehydrogenase (this −/− Irp2 cells (Fig. 2H and SI Appendix, Figs. S2A and S3A). This study), a burgeoning neuronal energy demand is hard to fulfill by reduction of Irp1 is presumably regulated by FBXL5, which can the remarkably weakened OXPHOS due to Irp2 ablation. These

Li et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 25, 2021 results suggest that the involvement of Irp2 in energy metabolism Materials and Methods is beyond direct iron regulation. MEFs derived from WT and global Irp2-deficient mice were generously given In conclusion, we demonstrated that Irp2 depletion increases by Dr. Tracey Rouault (Eunice Kennedy Shriver National Institute of Child the protein levels of Hif1α and Hif2α. Hif1α enhances aerobic Health and Human Development, NIH). Detailed information on cell lines −/− glycolysis in Irp2 MEFs by up-regulating target genes related and cell culture, antibodies and reagents, constructs and cell transfection, to the glycolytic pathway. Hif2α suppresses mitochondrial Western blot, determination of mitochondrial membrane potential, enzymatic activities, ATP and lactic acid contents, qRT-PCR, mitochondrial respi- OXPHOS, at least partially, by downregulating the expression of ration and glycolytic assays, and statistical analysis is available in SI Fxn and IscU to further reduce the biogenesis of mitochondrial Appendix, Materials and Methods. Fe–S and ETC subunits. These results indicate that Irp2 may switch energy metabolism between OXPHOS and glycolysis, ACKNOWLEDGMENTS. We thank Dr. Dong Wang for language assistance implying that high-energy-need tissues could be affected when and Dr. Xianwei Cui for technical assistance using the Seahorse Bioscience Irp2−/− XF24 Machine. This study was supported by grants from the National Basic Irp2 is deficient, as in mice, leading to neurological Research Program of China (Grant 2015CB856300) and by the National disorders (8–10). Natural Science Foundation of China (Grant 31571218).

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