Iron regulatory protein 2 modulates the switch fromaerobic glycolysis to oxidative phosphorylation inmouse embryonic fibroblastsHuihui 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 ofVascular 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) inmitochondrial iron homeostasis and function has been raised. Tounderstand how an IRP affects mitochondrial function, we usedglobally Irp2-depleted mouse embryonic fibroblasts (MEFs) andfound that Irp2 ablation significantly induced the expression ofboth hypoxia-inducible factor subunits, Hif1α and Hif2α. The in-crease of Hif1α up-regulated its targeted genes, enhancing glycol-ysis, and the increase of Hif2α down-regulated the expression ofiron–sulfur cluster (Fe–S) biogenesis-related and electron transportchain (ETC)-related genes, weakening mitochondrial respiration.Inhibition of Hif1α by genetic knockdown or a specific inhibitorprevented Hif1α-targeted gene expression, leading to decreasedaerobic glycolysis. Inhibition of Hif2α by genetic knockdown orselective disruption of the heterodimerization of Hif2α and Hif1βrestored the mitochondrial ETC and coupled oxidative phosphory-lation (OXPHOS) by enhancing Fe–S biogenesis and increasing ETC-related gene expression. Our results indicate that Irp2 modulatesthe metabolic switch from aerobic glycolysis to OXPHOS that ismediated by Hif1α and Hif2α in MEFs.

iron regulatory protein 2 | mitochondrial function | energy metabolism |hypoxia inducible factors

Iron is essential for growth and proliferation of mammaliancells due to its important roles in protein cofactors, hemes and

iron–sulfur clusters (Fe–S), which are involved in a number ofbiochemical pathways, including hemoglobin synthesis and themitochondrial respiration chain. Cellular iron homeostasis issecured by two orthologous iron regulatory proteins (IRPs),IRP1 and IRP2, both of which are iron-regulated RNA-bindingproteins that posttranscriptionally control the expression of aseries of iron-related genes, such as ferritin, transferrin receptor1 (TfR1), ferroportin 1 (FPN1), DMT1, and eALAS (1, 2). Whencells are iron-deficient in the labile pool, IRPs bind iron-responsive elements (IREs) located in the 5′-UTR of ferritinand FPN1 mRNA to inhibit its translation, which reduces ironstorage and export, and the IRE in the 3′-UTR of TfR1 andDMT1, which stabilizes the mRNA to facilitate iron import.When cells are iron-abundant, IRP1 is converted to a [4Fe-4S]-containing aconitase, and IRP2 is removed through iron-mediated proteasomal degradation (3, 4), which increases ferri-tin and FPN1 translation and promotes TfR1 and DMT1 mRNAdegradation, preventing additional iron absorption and avoidingexcess iron-induced injury.Studies have shown that mice lacking Irp2 have abnormal iron

contents in several tissues and develop microcytic anemia anderythropoietic protoporphyria (5, 6). Irp2 knockout mice alsohave symptoms of neurological disorders (7–9) due to thefunctional iron starvation in brain and spinal cord. This func-tional iron starvation is therefore considered to be causative ofthe impaired mitochondrial activity (10). However, the exactmechanism by which Irp2 sustains normal mitochondrial func-tion is still unclear.

In our previous study, we found that the Irp1- or Irp2-nullmutation in mouse embryonic fibroblasts (MEFs) caused de-creased expression of frataxin (Fxn) and iron–sulfur clusterscaffold protein IscU, two important components of the Fe–Sbiogenesis machinery (11). Deficiency of Fxn or IscU in humanand mouse cells limits mitochondrial function due to the lack ofsufficient Fe–S clusters (12, 13). Furthermore, IRP depletion-induced deficiency of Fxn and IscU specifically adversely af-fects the activity of the Fe–S-dependent mitochondrial re-spiratory chain, while the activities of other Fe–S-dependentenzymes, such as aconitase and xanthine dehydrogenase, areenhanced (11). Strangely, ATP is more highly produced inIrp2−/− MEFs than in WT (the present study). This resultseeming paradoxical to the low activity of the electron transportchain (ETC) and high content of ATP, suggesting a shift of themetabolic pathway in Irp2 ablation cells.Oxidative phosphorylation (OXPHOS) and glycolysis are two

key metabolic pathways for energy production. The switch fromone pathway to another is controlled by a number of factors,including two important transcription factors, HIF1 and HIF2.HIFs are heterogeneous dimers that are mainly composed ofan O2-labile alpha subunit (HIF1α or HIF2α) and a stablebeta subunit (HIF1β, also known as ARNT). The direct con-nection between Irp and Hif demonstrates that Hif2α is

Significance

Iron regulatory proteins (IRPs) control cellular iron homeostasis.Irp2 knockout mice show symptoms of neurological disorders,which are considered to result from impaired mitochondrialactivity. To explore the involvement of Irp2 in mitochondrialfunction, we examined the metabolic pathways of Irp2-depleted mouse embryonic fibroblasts. We found that Irp2deficiency switches cellular metabolic pathways from oxidativephosphorylation (OXPHOS) to aerobic glycolysis. We furtherrevealed that Irp2 deficiency induces the expression of Hif1αand Hif2α; Hif1α enhances aerobic glycolysis by upregulatingits target genes related to the glycolytic pathway, and Hif2αsuppresses mitochondrial Fe–S biosynthesis and OXPHOS. Thisidentified mechanism implies that high-energy-need tissues,such as the central nervous system, could be affected whenIrp2 is deficient, leading to neurological disorders.

Author contributions: H.L. and K.L. designed research; H.L., Y.L., L.S., J.C., J.W., W.Z., X.P.,and W.D. performed research; T.Q. and K.L. contributed new reagents/analytic tools; H.L.,T.Q., and K.L. analyzed data; and H.L. and K.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence should be addressed. Email: likuanyu@nju.edu.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1820051116/-/DCSupplemental.

Published online April 30, 2019.

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posttranscriptionally regulated by Irp1 through binding the IREin 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, whichmediates the up-regulation of erythropoietin (16–18). AlthoughHif2α 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 inwhich Irp2 is globally depleted, we demonstrated that increasedHif1α enhanced glycolysis by targeting a number of glycolyticpathway-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–Scluster assembly and by decreasing the expression of ETC sub-units and weakening OXPHOS. Therefore, Irp2 deficiencyswitches energy metabolism from OXPHOS to glycolysis.

ResultsIrp2 Ablation-Induced Mitochondrial Dysfunction Is Associated withthe Metabolic Switch from OXPHOS to Aerobic Glycolysis in MEFs.IRPs have been demonstrated to be important for mitochondrialiron supply and function (19). Consistent with this finding, weand other groups revealed in vivo and in vitro that a deficit ofavailable iron and reduction of mitochondrial Fe–S biogenesiscan be key factors in mitochondrial dysfunction in general (20)and in Irp2 ablated cells (10, 11). Here, we confirmed that theactivities of mitochondrial complexes I, II, and III significantlydecreased in Irp2−/− MEFs compared with WT (Fig. 1A), whichis consistent with the levels of the Fe–S-containing subunitsNdufs1 (of complex I), SdhB (of complex II), and Uqcrfs1 (ofcomplex III) in Irp2−/− cells (Fig. 1B and SI Appendix, Fig. S1). Inaddition, the amounts of other mitochondrial proteins, such ascytochrome C (CytC, an intermembrane protein) and ferroche-latase (Fech, a matrix protein), also decreased in Irp2−/− cells(Fig. 1B and SI Appendix, Fig. S1). To further detect any broadeffect of Irp2 deficiency on mitochondrial function, we measuredthe sensitivity of Irp2−/− cells to various inhibitors of mitochon-drial complexes. The results showed that the viability of Irp2−/− cellssignificantly decreased compared with WT (Fig. 1C), suggestingthat these cells are more sensitive to perturbations of mito-chondrial function after Irp2 deprivation. The mitochondrialmembrane potential (MMP), as measured using a specific andsensitive dye, JC-10, was much lower in Irp2−/− cells than inWT cells (Fig. 1D), suggesting that mitochondria in Irp2−/− cellsare more depolarized.Interestingly, although Irp2 deletion seriously weakened mi-

tochondrial function, the growth of Irp2−/− cells was not signifi-cantly retarded, and the difference between WT and mutant wasonly pronounced on the fourth day due to insufficient nutrients(Fig. 1E). Surprisingly, the level of ATP significantly increased inIrp2−/− cells compared with WT in the growth phase (day 2) (Fig.1F). We then speculated that aerobic glycolysis was enhanced toprovide enough ATP for cell growth. Therefore, we detected thelevels of several proteins involved in glycolysis, such as hexoki-nase 2 (HK2), glucose transporter 1 (Glut1), and lactate de-hydrogenase A/B (LdhA/B). The expression of these proteinswas significantly increased in Irp2−/− cells (Fig. 1G and SI Ap-pendix, Fig. S1). We also detected pyruvate dehydrogenase(Pdh), a vital regulatory enzyme that catalyzes the conversion ofpyruvate into acetyl-CoA and connects glycolysis to the TCAcycle. The protein level of Pdh was significantly reduced inIrp2−/− cells (Fig. 1G and SI Appendix, Fig. S1). However,phosphorylation of the E1α subunit of Pdh (p-Pdh–E1α(pSer232)), which leads to inactivation of the Pdh complex en-zymatic activity, was enhanced (Fig. 1G and SI Appendix, Fig.S1), suggesting that glycolytic metabolism was favored overmitochondria-dependent metabolism. To further verify these

results, we cultured cells in medium containing a high (4.5 g/L)or low (1.0 g/L) concentration of glucose for 3 d and measuredthe content of lactic acid, a by-product of the postglycolysispathway. As shown in Fig. 1H, Irp2−/− cells always produced andsecreted more lactic acid than WT cells under both glucoseconcentration conditions and in a concentration-dependentmanner. The protein levels of Hk2, Glut1, and LdhA/B allconsistently increased in Irp2−/− cells under a high glucose con-centration (Fig. 1I). We then evaluated cellular OXPHOS bydetecting the levels of related proteins. The expression of Pdh,Ndufs1, and Uqcrfs1 was not affected by different concentrationsof glucose in WT or Irp2−/− cells, although these proteins all

Fig. 1. Irp2 ablation-induced mitochondrial dysfunction is associated withenhanced aerobic glycolysis in MEFs. (A) Activities of ETC complexes in Irp2-deficient MEFs. CI, CII, and CIII, complexes I, II, and III. (B) Western blotanalysis of mitochondrial proteins, including Ndufs1 (a subunit of CI), SdhB (asubunit of CII), Uqcrfs1 (a subunit of CIII), Fech (a matrix enzyme ferroche-latase), CytC (an intermembrane space protein cytochrome C), and Cs (amatrix non-Fe–S citrate synthase). A representative image set is presented.Actin was used as a loading control. (C) Sensitivity of Irp2−/− cells to ETCcomplex inhibitors, including rotenone (10 μM, inhibitor of complex I),oxaloacetic acid (OAA) (100 μM, inhibitor of complex II), and antimycin A(10 μM, inhibitor of complex III). (D) MMP detected using JC-10. Greenfluorescence represents JC-10 monomers, and red fluorescence representsJC-10 aggregates. The ratio of red fluorescence to green fluorescence rep-resents the level of the mitochondrial membrane potential. (E) Growthcurves of WT and Irp2−/− cells. (F) Intracellular ATP content of WT and Irp2−/−

cells in the growth phase (day 2 after subculture). (G) A representative setfor proteins Hk2, Glut1, LdhA, LdhB, Pdh(-E1α), and p-Pdh(-E1α (pSer232))revealed by Western blot analysis. (H) Levels of medium lactic acid in WT andIrp2−/− cells cultured in medium containing 4.5 g/L glucose (H, high) or 1.0 g/Lglucose (L, low). (I) A representative set of Western blot analyses of glycolyticpathway-related proteins (Hk2, Glut1, LdhA, and LdhB) and oxidativephosphorylation pathway-related proteins (Pdh, Ndufs1, and Uqcrfs1). Actinwas used as a loading control. Representative blots from n = 3 experimentsare shown (each with duplicates). Values represent the mean ± SEM. One-way ANOVA (H) or Student’s t test (A, C, E, and F) was performed. *P < 0.05,**P < 0.01, ***P < 0.001, mutant vs. WT. ##P < 0.01, low vs. high concen-tration of glucose in medium.

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expressed less in Irp2−/− cells (Fig. 1I). These results stronglysuggest that Irp2 deficiency promotes cellular aerobic glycolysisand suppresses OXPHOS.To verify this hypothesis, the oxygen consumption rate (OCR)

and extracellular acidification rate (ECAR), as indicators ofmitochondrial respiration and the glycolytic rate, respectively,were measured using an Agilent Seahorse Analyzer. As illustratedin Fig. 2 A and B, Irp2−/− cells had lower resting OCR or OXPHOS

and a lower maximal mitochondrial capacity than WT cells aftertreatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone(FCCP), suggesting that Irp2−/− cells are less oxidative, consistentwith the lower mitochondria-derived ATP content. Simulta-neously, Irp2−/− cells had a much higher ECAR after glucose andoligomycin treatment (Fig. 2 C and D), suggesting that they aremore glycolytic. Collectively, these results demonstrated thatIrp2 deficiency induced a metabolic switch from OXPHOS toaerobic glycolysis.To further confirm that the metabolic switch was due to Irp2

deficiency, human IRP1 or IRP2, homologs of mouse Irp1 andIrp2, respectively, was expressed in Irp2-depleted MEFs to assessthe recovery of iron and energy metabolism. Cellular iron me-tabolism was evaluated (SI Appendix, Fig. S2), and the resultssupported the conserved iron regulatory function of human IRP1and IRP2. Evaluation of energy metabolism revealed that theexpression of IRP2 increased the activities of respiration com-plexes I, II, and III, while the expression of IRP1 in Irp2−/− cellsonly increased the activities of complexes I and II (Fig. 2E). Thisresult was in agreement with the sensitivity of Irp2−/− cells toinhibitors of mitochondrial ETC complexes (Fig. 2F). We alsofound that only IRP2, and not IRP1, significantly improved theMMP of Irp2−/− cells (Fig. 2G). Biochemical evidence revealedthat all of the tested OXPHOS-related protein levels, such asPdh, Ndufs1, and Uqcrfs1, significantly increased in Irp2−/− cellsafter IRP2 expression, but only the level of Pdh increased afterIRP1 expression (Fig. 2H and SI Appendix, Fig. S3). In-terestingly, the levels of the postglycolysis-related proteins LdhAand LdhB remained high after either IRP1 or IRP2 expression(Fig. 2H and SI Appendix, Fig. S3). However, the contents oflactic acid in both the cell lysate and medium of Irp2−/− cellswere reduced by IRP2 expression, not by IRP1 expression inmedium (Fig. 2I). Thus far, we have provided evidence that Irp2can shift cellular respiration in favor of OXPHOS over aerobicglycolysis.

Irp2 Absence-Induced Up-Regulation of Hif2α in MEFs AffectsMitochondrial Biogenesis. We further investigated the mecha-nism that drives the switch from OXPHOS to glycolysis in Irp2-deprived MEFs. HIF is a key transcriptional factor in regulatinga number of genes involved in glycolytic respiration, includingsome 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 Aand B, confirmed our hypothesis. We then treated Irp2−/− cellswith specific inhibitors of Hif1α (PX-478) and Hif2α (PT-2385).PX-478 transcriptionally and translationally inhibits Hif1α ex-pression, and PT-2385 selectively disrupts the heterodimeriza-tion of Hif2α with Hif1β, although their mechanisms of actionhave yet to be fully elucidated (see review in ref. 21). The con-centrations of the drugs were optimized (SI Appendix, Fig. S4C),and the effects of the inhibition were confirmed by the down-regulation of the Hif-targeted genes LdhA, endothelin 1 (Edn1),and Glut1 (Fig. 3B). The results suggested that LdhA and Glut1were mainly targeted by Hif1 and Edn1 was mainly targeted byHif2 in MEFs. Surprisingly, Fxn and IscU expression was sig-nificantly up-regulated by PT-2385 but not by PX-478 (SI Ap-pendix, Fig. S4C). We further examined a number of genesinvolved in iron metabolism and OXPHOS. Inhibition of Hif1αin Irp2−/− cells did not change the protein levels of iron-relatedgenes, such as Fxn and IscU, or mitochondrion-related genes,such as Pdh, Ndufs1, SdhB, and Uqcrfs1 (Fig. 3C and SI Ap-pendix, Fig. S5). By contrast, suppression of Hif2α in Irp2−/− cellssignificantly increased the protein levels of Pdh, Ndufs1, SdhB,Uqcrfs1, Fxn, and IscU (Fig. 3D and SI Appendix, Fig. S6). Theeffects of the specific inhibitors PX-478 and PT-2385 were also

Fig. 2. Human IRP2 rescues Irp2 ablation-induced mitochondrial dysfunctionand reverses energy metabolism in MEFs. (A) Profiles of the OCR in WT andIrp2−/− cells. Oligomycin, 1 μM; FCCP, 1 μM; rotenone/antimycin (Rot/AA),0.5 μM. (B) The calculated OCR for basal and maximal respiration and ATPproduction. (C) Profiles of the ECAR in WT and Irp2−/− cells.. Glucose, 10 mM;oligomycin, 1 μM; 2-deoxyglucose (2-DG), 50 mM. (D) The calculated ECAR forglycolysis and the glycolytic capacity. (E) Enzymatic activities of CI, CII, and CIIIdetermined in Irp2-deficient MEFs after transfection with pcMV-HA-IRP1 orpDEST-his-IRP2. (F) Sensitivities to ETC complex inhibitors after IRP1 or IRP2expression in Irp2−/− cells. The treatment with complex inhibitors was the sameas in Fig. 1. (G) MMP of Irp2−/− cells after expression of IRP1 or IRP2. *P = 0.0443;#P = 0.0274; NS, P = 0.0819. (H) Protein levels of IRP1, IRP2, LdhA, LdhB, Pdh,Ndufs1, and Uqcrfs1 determined byWestern blot analysis. (I) Levels of lactic acidin the medium or cell lysate of Irp2−/− cells after expression of IRP1 or IRP2.Actin was used as a loading control in Western blot analysis. Values representthe mean ± SEM (n = 3–5, each with duplicates). In B, D, E, F, G, and I, *P < 0.05,**P < 0.01, ***P < 0.001, mutant vs. WT; #P < 0.05, ##P < 0.01, ###P < 0.001, IRPrescue vs. nonrescue. NS, no significance, IRP1 rescue vs. nonrescue.

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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-dependentmetabolism.

Both Hif1 and Hif2 Collaboratively Mediate the Metabolic Switch ofIrp2−/− Cells. To further demonstrate whether the increased ex-pression of Hif1α and Hif2α induced by Irp2 deficiency was thecause of the cellular metabolic shift, we examined whether thephenotypes could be reversed after inhibition of Hif1α andHif2α. We measured the OCR and ECAR in Irp2−/− cells aftertreatment with PX-478 or PT-2385. The results showed thatbasal respiration, maximal respiration, and OXPHOS-dependentATP production were efficiently reversed by inhibiting Hif2α butnot by inhibiting Hif1α (Fig. 4 A and B). By contrast, both gly-colysis and the glycolysis capacity were significantly suppressedby inhibition of Hif1α but not by inhibition of Hif2α (Fig. 4 C andD). These results further validate that Irp2 deficiency enhancesglycolysis by inducing the expression of Hif1α and suppressesOXPHOS and mitochondrial biogenesis by inducing the expressionof Hif2α, thereby switching energy metabolism in MEFs.

To reveal the biochemical basis of the above observed phe-notypes, we measured the activities of mitochondrial ETCcomplexes I, II, and III and found that they were all significantlyincreased by inhibition of Hif2α alone or by inhibition of bothHif1α and Hif2α in Irp2−/− cells but not by inhibition of Hif1αalone (Fig. 4E). Although the TfR1 level remained constant for ironimport, ferritin expression was significantly increased (Fig. 4F), in linewith 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 de-termined by qRT-PCR (Left) and Western blot analysis (Right). (B) Effects ofHif1α and Hif2α inhibition by PX-478 (a specific inhibitor of Hif1α, 50 μM) andPT-2385 (a specific inhibitor of Hif2α, 50 μM), respectively, on their target genesLdhA, endothelin 1 (Edn1), and Glut1 in Irp2−/− cells. (C and D) Expression ofOXPHOS 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 Hif2αby PT-2385 (10–50 μM) (D) determined by Western blot analysis. DMSO as avehicle of PT-2385 was added at an identical volumewhen cells were treated. Arepresentative image set is presented, and the quantitative data of the proteinlevels are shown in the SI Appendix, Figs. S5 and S6. Values represent themean ± 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, nosignificance, with inhibitor vs. without inhibitor.

Fig. 4. Both Hif1α and Hif2α collaboratively mediate the metabolic switch ofIrp2−/− cells. (A) Profiles of the OCR, reflecting OXPHOS activity in Irp2−/− cellsafter treatment with PX-478 (50 μM) or PT-2385 (50 μM) for 24 h. (B) The cal-culated OCR for basal and maximal respiration and ATP production. (C) Profilesof the ECAR, reflecting glycolytic activity in Irp2−/− cells after treatment with PX-478 or PT-2385 for 24 h. (D) The calculated ECAR for glycolysis and glycolyticcapacity. (E) Activities of ETC complexes CI, CII, and CIII in Irp2−/− MEFs aftertreatment with PX-478, PT-2385, or both PX-478 and PT-2385 for 24 h. (F)Protein levels of a series of genes involved in either aerobic glycolysis orOXPHOS determined by Western blot analysis after inhibition of Hif1α and/orHif2α in Irp2−/− cells. (G) Levels of lactic acid in the medium of Irp2−/− MEFs aftertreatment with PX-478 and/or PT-2385 for 24 h. (H) Intracellular ATP content ofIrp2−/− cells after treatment with PX-478 and/or PT-2385. Values represent themean ± SEM (n = 3–5, each with duplicates). *P < 0.05, **P < 0.01, ***P < 0.001,mutant vs.WT; #P< 0.05, ##P< 0.01, ###P< 0.001, with inhibitor vs. without inhibitor.

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In accordance with this result, the expression of Fxn, IscU,Ndufs1, SdhB, and Uqcrfs1 significantly increased in Irp2−/− cellsafter inhibiting Hif2α, and the effects were even more profoundwith simultaneous inhibition of Hif1α and Hif2α (Fig. 4F). Theexpression of LdhA and HK2 was reduced (Fig. 4F) aftertreatment with PX-478, which was in agreement with thedrastically diminished production of lactic acid in the medium,while no change was observed after treatment with PT-2385alone (Fig. 4G). The combined treatment of PX-478 and PT-2385 showed an effect on the production of lactic acid similarto that with PX-478 alone (Fig. 4G). Furthermore, the ATPcontent correlated very well with the levels of lactic acid andincreased when Hif2 was inhibited (Fig. 4H). These resultsfurther prove that the effects of Hif1 and Hif2 are independentand that aerobic glycolysis is a major metabolic pathway inIrp2−/− MEFs.The effects of Hif1α and Hif2α on the metabolic switch were

further validated by a shRNA or siRNA knockdown approach.The knockdown efficiency was first evaluated (SI Appendix, Fig.S9 A and B), and the best shRNA and siRNA were used to knockdown Hif1α and Hif2α, respectively. The Western blot resultsshowed that the expression of the Hif1-targeted genes HK2 andLdhA was significantly reduced after Hif1α was knocked downwith siRNA (SI Appendix, Fig. S9C). The consequence was thereduced production of lactic acid (SI Appendix, Fig. S9D). Sim-ilarly, the expression of Hif2-targeted genes was reduced whenHif2α was knocked down with shRNA (SI Appendix, Fig. S9E).In line with the drug inhibition of Hif2, Fe–S biogenesis-related(Fxn and IscU) and OXPHOS-related (complex I subunitNdufs1, complex II subunit SdhB, and complex III subunitUqcrfs1) genes were up-regulated (SI Appendix, Fig. S9F). Asanticipated, the ATP content increased further (SI Appendix,Fig. S9G). Combining the results from the drug inhibition andgenetic approaches, we concluded that the Irp2 ablation-induced metabolic switch is mediated by up-regulated Hif1αand Hif2α.

DiscussionIn this study, we first found that Irp2-deficient MEFs had nearlynormal growth despite their significantly low ETC activity. In-terestingly, the ATP content was relatively higher in mutant cells.We further discovered that Irp2−/− cells favored aerobic glycol-ysis over OXPHOS, which was triggered by up-regulated Hif1αand Hif2α. Inhibition of both Hifs suppressed aerobic glycolysisand enhanced OXPHOS, thereby switching respiration fromaerobic glycolysis to OXPHOS in Irp2−/− cells (illustrated in SIAppendix, Fig. S10). This illustration is in line with previousstudies, in which Hif1 and Hif2, while sharing structural simi-larity and common target genes, have unique targets involved indifferent pathways (22, 23).Under normal oxygen conditions, prolyl hydroxylase domain

enzymes (PHDs), which are master regulators of the hypoxiaresponse (24), hydroxylate HIFα subunits at conserved prolines,leading to HIFα degradation by the proteasome. PHD activityrequires iron binding at an active site. Therefore, Hif1α is sta-bilized by iron starvation, which is induced by Irp2 ablation in thisstudy. Hif1α contains a unique transactivation domain that al-lows preferential activation of hypoxia-responsive glycolyticgenes; its downstream genes, such as HK2, Glut1, and LdhA, areup-regulated in Irp2−/− cells. This result was proven by the ad-dition of iron, which reduced Hif1α protein levels (SI Appendix,Fig. S11). However, up-regulated Hif2α did not respond to irontreatment (SI Appendix, Figs. S11A and S12B). Hif2α stabiliza-tion seems more complex because it can be regulated both byiron, similar to Hif1α, and directly by Irps (15, 25), probablymainly by Irp1 (14). Interestingly, the Irp1 levels were reduced inIrp2−/− cells (Fig. 2H and SI Appendix, Figs. S2A and S3A). Thisreduction of Irp1 is presumably regulated by FBXL5, which can

target both IRP1 and IRP2 for degradation (4, 26). A stronginduction of FBXL5 was observed when the cytosolic Fe–S as-sembly system was impaired, which contributed to the degrada-tion of the Irp1 protein (26). This degradation likely results inHif2α up-regulation in Irp2−/− cells, in line with the unalteredprotein level of Hif2α after the addition of iron (SI Appendix,Fig. S11). These results suggest that Irp1 reduction rather thaniron starvation is, at least partially, causative of Hif2α stabiliza-tion in Irp2−/− cells. This result was verified by exogenous ex-pression of IRP1 in Irp2−/− cells, which reversed the Hif2αprotein levels to some extent (SI Appendix, Fig. S2), very likelythrough IRP1–IRE binding to the 5′-UTR of Hif2α mRNA toinhibit translation.Inhibition of Hif1α in Irp2−/− cells suppressed the Hif1α target

genes HK2, Glut1, and LdhA. As a result, aerobic glycolysiswas repressed, and the lactic acid levels decreased, whereasOXPHOS-related genes and enzyme activities did not respond toHif1α inhibition. Thus, the decreased ATP content caused byHif1α inhibition is glycolysis-dependent. By contrast, Hif2α in-hibition in Irp2−/− cells drastically up-regulated the OXPHOS-related genes Ndufs1, SdhB, and Uqcrfs1, leading to the resto-ration of the activities of complexes I–III. Meanwhile, the ATPcontent increased further, proving that Irp2 depletion-inducedHif2α represses OXPHOS and reduces mitochondrion-dependentATP production. This effect is likely attributed to the suppres-sion of IscU and Fxn (this study and refs. 11 and 27). IscU hasbeen revealed to be a member of the miR-210 regulon duringhypoxia and adversely controls mitochondrial metabolism (28).The promoter of miR-210 contains a hypoxic responsive element(HRE) for Hif binding (29). Therefore, down-regulation of IscUin Irp2−/− MEFs is presumably through the miR-210–Hif axis.Hif1 and Hif2, both in combination (30) and individually (31),have been verified to target miR-210 in various tumor cells.Remarkably, in Irp2−/− MEFs, only the inhibition of Hif2α, notthat of Hif1α, increased IscU expression, suggesting that miR-210 is regulated by Hif2 in MEFs. Strikingly, Fxn exhibits verysimilar responses to Irp2 depletion (ref. 11 and this study), Hifinhibition (this study), and iron regulation (12, 32) as IscU.Human FXN has been reported to be directly regulated by Hif1,not by Hif2 (33). Mouse Fxn, in contrast, is controlled by Hif2,not by Hif1 (34). The up-regulation of FXN by both Hifs isthought to occur through the binding of HIF-HRE to the promoterregion of FXN. However, we found that Hif2, but not Hif1, down-regulated the expression of Fxn in MEFs. The mechanism needsto be investigated further.Consistently, we found here and previously (11) that Irp1 and

Irp2 had distinct impacts on mitochondrial metabolism, althoughthey are interchangeable in terms of iron metabolism. The rescueexperiments (Fig. 2E) showed that either IRP1 or IRP2 couldreverse the Irp2 deficiency-induced enzymatic defects of com-plexes I and II, whereas the activity of complex III could only bereversed by IRP2 expression. Comparable results have beenreported in which tempol treatment restored complex I activityof Irp2−/− mice by converting Irp1 from the cytosolic aconitase tothe IRE binding form for iron uptake to improve the neurode-generative symptoms of Irp2−/− mice (35). Tempol-induced ironbioavailability, presumably, also reduces Hif1α stability. Theimportant role of Hif1α in neurodegenerative diseases has beenproposed, where Hif1α can be considered to be a therapeutictarget (36). Moreover, inhibition of Hif1α blocked glycolysis,which is the main metabolic pathway to generate ATP and lacticacid in Irp2−/− cells. The high level of lactic acid or methyl-glyoxal, a highly reactive dicarbonyl compound inevitably formedas a by-product of glycolysis, might be toxic to neuronal function(37, 38). Despite the astrocyte-neuron lactate shuttle hypothesis(39) and the high expression of lactate dehydrogenase (thisstudy), a burgeoning neuronal energy demand is hard to fulfill bythe remarkably weakened OXPHOS due to Irp2 ablation. These

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results suggest that the involvement of Irp2 in energy metabolismis beyond direct iron regulation.In conclusion, we demonstrated that Irp2 depletion increases

the protein levels of Hif1α and Hif2α. Hif1α enhances aerobicglycolysis in Irp2−/− MEFs by up-regulating target genes relatedto the glycolytic pathway. Hif2α suppresses mitochondrialOXPHOS, at least partially, by downregulating the expression ofFxn and IscU to further reduce the biogenesis of mitochondrialFe–S and ETC subunits. These results indicate that Irp2 mayswitch energy metabolism between OXPHOS and glycolysis,implying that high-energy-need tissues could be affected whenIrp2 is deficient, as in Irp2−/− mice, leading to neurologicaldisorders (8–10).

Materials and MethodsMEFs derived from WT and global Irp2-deficient mice were generously givenby Dr. Tracey Rouault (Eunice Kennedy Shriver National Institute of ChildHealth and Human Development, NIH). Detailed information on cell linesand cell culture, antibodies and reagents, constructs and cell transfection,Western blot, determination of mitochondrial membrane potential, enzy-matic activities, ATP and lactic acid contents, qRT-PCR, mitochondrial respi-ration and glycolytic assays, and statistical analysis is available in SIAppendix, Materials and Methods.

ACKNOWLEDGMENTS. We thank Dr. Dong Wang for language assistanceand Dr. Xianwei Cui for technical assistance using the Seahorse BioscienceXF24 Machine. This study was supported by grants from the National BasicResearch Program of China (Grant 2015CB856300) and by the NationalNatural Science Foundation of China (Grant 31571218).

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