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Mitophagy mediates metabolic reprogramming of induced pluripotent stem cells undergoing endothelial differentiation

2021; Elsevier BV; Volume: 297; Issue: 6 Linguagem: Inglês

10.1016/j.jbc.2021.101410

1083-351X

Sarah Krantz, Young-Mee Kim, Shubhi Srivastava, Joseph W. Leasure, Péter T. Tóth, Glenn Marsboom, Jalees Rehman,

Mitochondrial Function and Pathology

Pluripotent stem cells are known to shift their mitochondrial metabolism upon differentiation, but the mechanisms underlying such metabolic rewiring are not fully understood. We hypothesized that during differentiation of human induced pluripotent stem cells (hiPSCs), mitochondria undergo mitophagy and are then replenished by the biogenesis of new mitochondria adapted to the metabolic needs of the differentiated cell. To evaluate mitophagy during iPSC differentiation, we performed live cell imaging of mitochondria and lysosomes in hiPSCs differentiating into vascular endothelial cells using confocal microscopy. We observed a burst of mitophagy during the initial phases of hiPSC differentiation into the endothelial lineage, followed by subsequent mitochondrial biogenesis as assessed by the mitochondrial biogenesis biosensor MitoTimer. Furthermore, hiPSCs undergoing differentiation showed greater mitochondrial oxidation of fatty acids and an increase in ATP levels as assessed by an ATP biosensor. We also found that during mitophagy, the mitochondrial phosphatase PGAM5 is cleaved in hiPSC-derived endothelial progenitor cells and in turn activates β-catenin-mediated transcription of the transcriptional coactivator PGC-1α, which upregulates mitochondrial biogenesis. These data suggest that mitophagy itself initiates the increase in mitochondrial biogenesis and oxidative metabolism through transcriptional changes during endothelial cell differentiation. In summary, these findings reveal a mitophagy-mediated mechanism for metabolic rewiring and maturation of differentiating cells via the β-catenin signaling pathway. We propose that such mitochondrial-nuclear cross talk during hiPSC differentiation could be leveraged to enhance the metabolic maturation of differentiated cells. Pluripotent stem cells are known to shift their mitochondrial metabolism upon differentiation, but the mechanisms underlying such metabolic rewiring are not fully understood. We hypothesized that during differentiation of human induced pluripotent stem cells (hiPSCs), mitochondria undergo mitophagy and are then replenished by the biogenesis of new mitochondria adapted to the metabolic needs of the differentiated cell. To evaluate mitophagy during iPSC differentiation, we performed live cell imaging of mitochondria and lysosomes in hiPSCs differentiating into vascular endothelial cells using confocal microscopy. We observed a burst of mitophagy during the initial phases of hiPSC differentiation into the endothelial lineage, followed by subsequent mitochondrial biogenesis as assessed by the mitochondrial biogenesis biosensor MitoTimer. Furthermore, hiPSCs undergoing differentiation showed greater mitochondrial oxidation of fatty acids and an increase in ATP levels as assessed by an ATP biosensor. We also found that during mitophagy, the mitochondrial phosphatase PGAM5 is cleaved in hiPSC-derived endothelial progenitor cells and in turn activates β-catenin-mediated transcription of the transcriptional coactivator PGC-1α, which upregulates mitochondrial biogenesis. These data suggest that mitophagy itself initiates the increase in mitochondrial biogenesis and oxidative metabolism through transcriptional changes during endothelial cell differentiation. In summary, these findings reveal a mitophagy-mediated mechanism for metabolic rewiring and maturation of differentiating cells via the β-catenin signaling pathway. We propose that such mitochondrial-nuclear cross talk during hiPSC differentiation could be leveraged to enhance the metabolic maturation of differentiated cells. Pluripotent stem cells such as human induced pluripotent stem cells (hiPSCs) rely mainly on glycolysis for cellular ATP (1Varum S. Momcilovic O. Castro C. Ben-Yehudah A. Ramalho-Santos J. Navara C.S. Enhancement of human embryonic stem cell pluripotency through inhibition of the mitochondrial respiratory chain.Stem Cell Res. 2009; 3: 142-156Crossref PubMed Scopus (132) Google Scholar, 2Folmes C.D. Nelson T.J. Martinez-Fernandez A. Arrell D.K. Lindor J.Z. Dzeja P.P. Ikeda Y. Perez-Terzic C. Terzic A. Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming.Cell Metab. 2011; 14: 264-271Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar, 3Panopoulos A.D. Yanes O. Ruiz S. Kida Y.S. Diep D. Tautenhahn R. Herrerias A. Batchelder E.M. Plongthongkum N. Lutz M. Berggren W.T. Zhang K. Evans R.M. Siuzdak G. Izpisua Belmonte J.C. The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming.Cell Res. 2012; 22: 168-177Crossref PubMed Scopus (380) Google Scholar) and use glutamine metabolism to sustain pluripotency (4Marsboom G. Zhang G.F. Pohl-Avila N. Zhang Y. Yuan Y. Kang H. Hao B. Brunengraber H. Malik A.B. Rehman J. Glutamine metabolism regulates the pluripotency transcription factor OCT4.Cell Rep. 2016; 16: 323-332Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 5Palm W. Thompson C.B. Nutrient acquisition strategies of mammalian cells.Nature. 2017; 546: 234-242Crossref PubMed Scopus (198) Google Scholar). However, when stem cells differentiate, their metabolism also shifts toward enhanced oxidative phosphorylation and mitochondrial ATP generation (6Mathieu J. Ruohola-Baker H. Metabolic remodeling during the loss and acquisition of pluripotency.Development. 2017; 144: 541-551Crossref PubMed Scopus (96) Google Scholar). Importantly, the metabolic shift can reinforce cell differentiation (7Chung S. Arrell D.K. Faustino R.S. Terzic A. Dzeja P.P. Glycolytic network restructuring integral to the energetics of embryonic stem cell cardiac differentiation.J. Mol. Cell Cardiol. 2010; 48: 725-734Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), thus suggesting there is a positive feedback loop between cell differentiation and metabolic rewiring. One such example of the cross talk between cell metabolism and the pluripotency state occurs when pluripotent cells downregulate glutamine metabolism, which in turn increases ROS generation and reduces activity of the pluripotency transcription factor OCT4, thus further stabilizing differentiation and the shift away from pluripotency (4Marsboom G. Zhang G.F. Pohl-Avila N. Zhang Y. Yuan Y. Kang H. Hao B. Brunengraber H. Malik A.B. Rehman J. Glutamine metabolism regulates the pluripotency transcription factor OCT4.Cell Rep. 2016; 16: 323-332Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The recognition that metabolic reprogramming is an essential feature of stem cell differentiation (8Esteban-Martinez L. Boya P. BNIP3L/NIX-dependent mitophagy regulates cell differentiation via metabolic reprogramming.Autophagy. 2018; 14: 915-917Crossref PubMed Scopus (56) Google Scholar, 9Zheng X. Boyer L. Jin M. Mertens J. Kim Y. Ma L. Ma L. Hamm M. Gage F.H. Hunter T. Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation.Elife. 2016; 5e13374Crossref PubMed Scopus (269) Google Scholar) raises the important question of how mitochondria in pluripotent stem cells are reprogrammed during differentiation. Mitophagy (the autophagy of mitochondria) is the selective elimination of depolarized or damaged mitochondria. Generally, mitophagy occurs as a method of quality control, eliminating damaged mitochondria with mutant mtDNA or misfolded proteins in order to maintain mitochondrial health (10Esteban-Martinez L. Sierra-Filardi E. McGreal R.S. Salazar-Roa M. Marino G. Seco E. Durand S. Enot D. Grana O. Malumbres M. Cvekl A. Cuervo A.M. Kroemer G. Boya P. Programmed mitophagy is essential for the glycolytic switch during cell differentiation.EMBO J. 2017; 36: 1688-1706Crossref PubMed Scopus (166) Google Scholar, 11Ng M.Y.W. Wai T. Simonsen A. Quality control of the mitochondrion.Dev. Cell. 2021; 56: 881-905Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The canonical pathway for mitophagy involves the serine/threonine kinase PINK1 (PTEN-induced kinase 1) and the E3 ubiquitin ligase Parkin (12Palikaras K. Lionaki E. Tavernarakis N. Mechanisms of mitophagy in cellular homeostasis, physiology and pathology.Nat. Cell Biol. 2018; 20: 1013-1022Crossref PubMed Scopus (476) Google Scholar). PINK1 continuously cycles from the cytosol to the mitochondrial outer membrane. If the mitochondria are healthy, PINK1 is cleaved and is released back into the cytosol (13Vives-Bauza C. Zhou C. Huang Y. Cui M. de Vries R.L. Kim J. May J. Tocilescu M.A. Liu W. Ko H.S. Magrane J. Moore D.J. Dawson V.L. Grailhe R. Dawson T.M. et al.PINK1-dependent recruitment of Parkin to mitochondria in mitophagy.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 378-383Crossref PubMed Scopus (1189) Google Scholar). When mitochondria are damaged and the mitochondrial membrane is depolarized, then PINK1 is stabilized and accumulates at the membrane. PINK1 phosphorylates the mitochondrial outer membrane protein Mitofusin 2, which in turn serves as a receptor for Parkin (14Chen Y. Dorn 2nd., G.W. PINK1-phosphorylated mitofusin 2 is a Parkin receptor for culling damaged mitochondria.Science. 2013; 340: 471-475Crossref PubMed Scopus (836) Google Scholar). Parkin then translocates to the mitochondria to ubiquitinate multiple proteins and trigger autophagy mechanisms to remove the mitochondria (13Vives-Bauza C. Zhou C. Huang Y. Cui M. de Vries R.L. Kim J. May J. Tocilescu M.A. Liu W. Ko H.S. Magrane J. Moore D.J. Dawson V.L. Grailhe R. Dawson T.M. et al.PINK1-dependent recruitment of Parkin to mitochondria in mitophagy.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 378-383Crossref PubMed Scopus (1189) Google Scholar, 15Matsuda N. Sato S. Shiba K. Okatsu K. Saisho K. Gautier C.A. Sou Y.S. Saiki S. Kawajiri S. Sato F. Kimura M. Komatsu M. Hattori N. Tanaka K. PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy.J. Cell Biol. 2010; 189: 211-221Crossref PubMed Scopus (1263) Google Scholar). While mitophagy has primarily been studied in the setting of culling damaged mitochondria in the setting of injury or disease, we posited that mitophagy may serve as a physiologic mechanism for the metabolic rewiring of differentiating stem cells by removing mitochondria adapted to the pluripotent state and replacing them with new mitochondria configured for the differentiated cell state. The vast majority of mitochondrial proteins are encoded by the nuclear genome; therefore, any initiation of compensatory mitochondrial biogenesis in response to mitophagy would require communication between mitochondria and the nucleus. One such potential mediator is the mitochondrial protein PGAM5 (Phosphoglycerate Mutase Family Member 5), a serine/threonine phosphatase that is embedded in the mitochondrial outer membrane (16Jedrzejas M.J. Structure, function, and evolution of phosphoglycerate mutases: Comparison with fructose-2,6-bisphosphatase, acid phosphatase, and alkaline phosphatase.Prog. Biophys. Mol. Biol. 2000; 73: 263-287Crossref PubMed Scopus (114) Google Scholar) and is cleaved and released into the cytosol during mitophagy (17Bernkopf D.B. Jalal K. Bruckner M. Knaup K.X. Gentzel M. Schambony A. Behrens J. Pgam5 released from damaged mitochondria induces mitochondrial biogenesis via Wnt signaling.J. Cell Biol. 2018; 217: 1383-1394Crossref PubMed Scopus (42) Google Scholar, 18Sekine S. Kanamaru Y. Koike M. Nishihara A. Okada M. Kinoshita H. Kamiyama M. Maruyama J. Uchiyama Y. Ishihara N. Takeda K. Ichijo H. Rhomboid protease PARL Mediates the mitochondrial membrane potential loss-induced cleavage of PGAM5.J. Biol. Chem. 2012; 287: 34635-34645Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). It enhances signaling via the β-catenin pathway by dephosphorylating β-catenin, thus stabilizing it and allowing it to translocate to the nucleus to transcribe Wnt/β-catenin pathway genes (17Bernkopf D.B. Jalal K. Bruckner M. Knaup K.X. Gentzel M. Schambony A. Behrens J. Pgam5 released from damaged mitochondria induces mitochondrial biogenesis via Wnt signaling.J. Cell Biol. 2018; 217: 1383-1394Crossref PubMed Scopus (42) Google Scholar). In this study, we show that mitophagy and subsequent mitochondrial biogenesis are required for the differentiation of hiPSCs to endothelial cells through the interaction of cleaved PGAM5 and β-catenin. Furthermore, we show that PGAM5 cleavage leads to differentiation of progenitor cells into more mature endothelial cells and the biogenesis of new mitochondria that exhibit a shift from mitochondrial glutamine metabolism to fatty acid metabolism. This novel feedback mechanism of mitochondrial-nuclear cross talk in differentiating pluripotent stem cells provides new insights into the intersection of metabolic and developmental signaling. To investigate whether mitophagy is activated during differentiation, we first differentiated hiPSCs to ECs (endothelial cells) using a three-step protocol (Fig. 1A). Cells were initially differentiated toward a mesodermal lineage, then an endothelial progenitor stage, and lastly to a mature differentiated endothelial state in which the cells express the endothelial adherens junction molecule VE-Cadherin (Fig. 1B). iPSC-ECs represent only VE-Cadherin positive cells. To further characterize iPSC-ECs, two iPSC lines (273 and C2) were differentiated to ECs, and the iPSC-EC identity was seen in the form of cobblestone morphology (Fig. S1A), as well as flow cytometry (Fig. S1B), which showed that more than 90% of cells express the cell surface proteins VE-Cadherin and CD31. A capillary-like tube formation assay was performed (Fig. S1C), which demonstrated that iPSC-ECs but not iPSCs are able to form a vascular-like network. Moreover, mRNA levels of VE-Cadherin, CD31, and VEGFR2 (Fig. S1D) showed a significant increase in these markers. Together these results show that iPSC-ECs in this study exhibit endothelial cell traits. We used this endothelial differentiation approach because human pluripotent stem cells demonstrate a clear metabolic shift characterized by downregulation of mitochondrial glutamine metabolism when pluripotent stem cells differentiate into mature endothelial cells (4Marsboom G. Zhang G.F. Pohl-Avila N. Zhang Y. Yuan Y. Kang H. Hao B. Brunengraber H. Malik A.B. Rehman J. Glutamine metabolism regulates the pluripotency transcription factor OCT4.Cell Rep. 2016; 16: 323-332Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). To determine whether mitophagy occurs during differentiation, the cells at the defined differentiation stages were stained with MitoTracker (green) and Lysotracker (red), followed by confocal microscopy. Mitophagy was evaluated by quantifying colocalization of lysosomes and mitochondria (Fig. 1, C and D). Mitophagy increased early in differentiation (at days 2 and 4) and then returned to baseline after cells reached the differentiated EC stage at day 7. We also examined the change in Ser443 Phospho-Mfn2 and PINK1, key mediators of the canonical PINK1/Parkin/Mitofusin 2 pathway. As shown in Figure 1, E–G, Phospho-Mfn2 and PINK1 protein levels increased on days 2 and 4, concomitantly with the observed increase in mitophagy, suggesting that the canonical mitophagy pathway was activated during differentiation of hiPSCs. To understand whether the increase in mitophagy during differentiation was also seen during differentiation into other cell lineages, we differentiated two iPSC cell lines (273 and C2) to cardiomyocytes using a 15-day cardiomyocyte (CM) differentiation protocol (19Kwon Y. Nukala S.B. Srivastava S. Miyamoto H. Ismail N.I. Jousma J. Rehman J. Ong S.B. Lee W.H. Ong S.G. Detection of viral RNA fragments in human iPSC cardiomyocytes following treatment with extracellular vesicles from SARS-CoV-2 coding sequence overexpressing lung epithelial cells.Stem Cell Res. Ther. 2020; 11: 514Crossref PubMed Scopus (23) Google Scholar). We used Mitotracker (green) and Lysotracker (red) colabeling to assess mitophagy on day 2, day 5, and day 9. While the observed increase in mitophagy was not as prominent as during endothelial differentiation, there was a significant increase in mitophagy early on at day 2 (Fig. S2, A and B), corresponding to increases in the mitophagy mediators PINK1 and Phospho-Mfn2 (Fig. S2, C and D). To determine whether mitophagy was followed by compensatory mitochondrial biogenesis to replenish the culled mitochondria, we utilized the doxycycline inducible MitoTimer construct (20Hernandez G. Thornton C. Stotland A. Lui D. Sin J. Ramil J. Magee N. Andres A. Quarato G. Carreira R.S. Sayen M.R. Wolkowicz R. Gottlieb R.A. MitoTimer: A novel tool for monitoring mitochondrial turnover.Autophagy. 2013; 9: 1852-1861Crossref PubMed Scopus (100) Google Scholar). In cells expressing the doxycycline-inducible fluorescent protein construct MitoTimer, mitochondrial biogenesis can be quantified as a ratio of fluorescence emission spectra. MitoTimer in newly synthesized mitochondria exhibit green fluorescence but over time, aging mitochondria have increased red fluorescence (Fig. 2A). MitoTimer is used to track mitochondrial aging and biogenesis. We examined whether mitophagy in differentiating hiPSCs was followed by mitochondrial biogenesis subsequent to the early mitophagy during differentiation. Using this MitoTimer construct, we compared control hiPSCs with cells differentiated for 4 days. We first stimulated the cells with a pulse of 2 μg/ml doxycycline for 24 h. After 48 h, all the mitochondria were red fluorescent indicative of mitochondrial aging. The cells were given a second pulse of doxycycline for the 24 h of day 4 differentiation, and then newly synthesized mitochondria were evaluated by monitoring green mitochondria using confocal microscopy in fixed cells. De novo mitochondrial biogenesis was analyzed as the ratio of green over red fluorescence intensity. We found a doubling of newly synthesized mitochondria in day four differentiated endothelial progenitor cells as compared with undifferentiated hiPSCs indicating more de novo mitochondrial biogenesis after the initial mitophagy (Fig. 2, B and C). We next assessed PGC-1α, a key regulator of mitochondrial biogenesis, in differentiating hiPSCs. On day 4 of endothelial differentiation, there is a significant increase in PGC1α expression (Fig. 2D). To visualize this increase in mitochondrial mass, we fixed cells at defined stages of differentiation and stained for TOMM20 (Fig. 2, E and F). Interestingly, we found that mitochondrial mass steadily increases after the initial mitophagy phase, suggesting that the de novo mitochondrial biogenesis outpaces mitophagy. We also examined PGC-1α mRNA levels during cardiomyocyte differentiation and found that the levels also increase following mitophagy (Fig. S2E), concurrent with an increase in the cardiomyocyte markers TNNT2 and RYR2 (Fig. S2F), which suggests that cardiomyocytes increase mitochondrial mass while differentiating into cardiomyocytes. We next examined whether the axis of mitophagy- mitochondrial biogenesis directs a metabolic shift in the differentiation of hiPSCs to iPSC-ECs. We used the fluorescence ATP sensor PercevalHR to quantify the ratio of ATP to ADP (21Tantama M. Martinez-Francois J.R. Mongeon R. Yellen G. Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio.Nat. Commun. 2013; 4: 2550Crossref PubMed Scopus (235) Google Scholar). Interestingly, we found that ATP production was significantly increased in hiPSCs differentiating to endothelial progenitor (day 4) cells (Fig 3A, left panel and Fig. 3B) concomitant with the increase in mitochondrial biogenesis. This suggests that upon differentiation of hiPSCs to endothelial progenitor cells (day 4), the cells are able to generate greater amounts of ATP as compared with undifferentiated iPSCs. We next tested whether suppressing mitophagy by depletion of Mfn2 (Fig. 3C) would impact the metabolic function of differentiating hiPSCs. hiPSCs were infected with lentiviral doxycycline inducible Mfn2 shRNA construct to prevent mitophagy. The efficacy of this approach to decrease Mfn2-mediated mitophagy was confirmed using mitophagy assessment by Lysotracker and Mitotracker. iPSCs with and without Mitofusin 2 knockdown were treated with the mitophagy inducers oligomycin and antimycin (O/A). We observed a marked attenuation of mitophagy induction in Mfn2 depleted cells (Fig. S3A) indicating that Mfn2 is critical for mitophagy to occur in iPSCs. As shown in Figure 3, A and B, Mfn2-depleted endothelial progenitor cells at day 4 have significantly decreased ATP production compared with control cells. These data suggest that mitophagy regulates metabolic activity by activating the production of ATP. Next, we determined whether the iPSC-ECs changed metabolism as compared with the parental iPSCs. Human stem cells rely on glutamine metabolism for their metabolic needs (4Marsboom G. Zhang G.F. Pohl-Avila N. Zhang Y. Yuan Y. Kang H. Hao B. Brunengraber H. Malik A.B. Rehman J. Glutamine metabolism regulates the pluripotency transcription factor OCT4.Cell Rep. 2016; 16: 323-332Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 22Carey B.W. Finley L.W. Cross J.R. Allis C.D. Thompson C.B. Intracellular alpha-ketoglutarate maintains the pluripotency of embryonic stem cells.Nature. 2015; 518: 413-416Crossref PubMed Scopus (547) Google Scholar). We therefore measured the mRNA levels of 2 K and L isoforms of glutaminase, which converts glutamine to glutamate using RT-qPCR. The levels of both glutaminases markedly decreased in differentiated ECs (Fig. 3, D and E) indicating a shift away from glutamine metabolism. We also investigated fatty acid oxidation which mature ECs use to provide acetyl-CoA to the TCA cycle (23Schoors S. Bruning U. Missiaen R. Queiroz K.C. Borgers G. Elia I. Zecchin A. Cantelmo A.R. Christen S. Goveia J. Heggermont W. Godde L. Vinckier S. Van Veldhoven P.P. Eelen G. et al.Fatty acid carbon is essential for dNTP synthesis in endothelial cells.Nature. 2015; 520: 192-197Crossref PubMed Scopus (341) Google Scholar, 24Falkenberg K.D. Rohlenova K. Luo Y. Carmeliet P. The metabolic engine of endothelial cells.Nat. Metab. 2019; 1: 937-946Crossref PubMed Scopus (34) Google Scholar). In contrast to the glutaminases, Fatty Acid Binding Protein 4 (FABP4), a cytosolic protein that binds fatty acids, is significantly increased in iPSC-ECs but not in hiPSCs (Fig. 3F) We also observed a similar upregulation for CPT1A, an essential mitochondrial enzyme involved in fatty acid beta-oxidation (Fig. 3G). We also tested this metabolic shift in another iPSC line (C2 cells) (Fig. S3, B–D) and found a similar decrease in glutamine metabolism gene expression by showing mRNA levels of K-Glutaminase and L-Glutaminase and corresponding upregulation of the mRNA levels of the fatty acid metabolism regulator FABP4. Moreover, to assess the metabolic shift in iPSC-ECs, we measured the mitochondrial oxygen consumption rate (OCR) using the Seahorse Analyzer (Fig. 3H). At basal levels, iPSC-ECs had a significantly greater basal respiration and maximal respiratory capacity than iPSCs (Fig. 3, I and J) indicating increased utilization of mitochondrial respiration. There was also an increase in OCR in iPSC-ECs after the addition of the fatty acid palmitate and carnitine (for mitochondrial transport of the palmitate). However, this increase is not present in iPSCs suggesting that mitochondria in iPSC-ECs were able to increasingly utilize fatty acids as a fuel when compared with undifferentiated iPSCs. We next investigated the underlying mechanism of how mitophagy could induce the transcriptional programs involved in mitochondrial biogenesis. One way that the mitochondria can communicate with transcriptional programs is through the mitochondrial phosphatase PGAM5. This protein can be cleaved upon mitophagy and dephosphorylates β-catenin, which triggers its translocation to the nucleus to activate Wnt target genes (17Bernkopf D.B. Jalal K. Bruckner M. Knaup K.X. Gentzel M. Schambony A. Behrens J. Pgam5 released from damaged mitochondria induces mitochondrial biogenesis via Wnt signaling.J. Cell Biol. 2018; 217: 1383-1394Crossref PubMed Scopus (42) Google Scholar). We first examined whether this PGAM5 cleavage occurs in mitophagy induced-iPSC differentiation. iPSCs were treated with the mitophagy inducers oligomycin/antimycin for 3 h. We found that PGAM5 is indeed cleaved as shown through an increase of the bottom (cleaved) band over the top (full) band of PGAM5 (Fig. 4, A and B). We next investigated if PGAM5 is cleaved during differentiation. While mitophagy is increased on days 2 and 4, we found an increase in cleaved PGAM5 only on day 4 (Fig. 4, C and D), which is also the day that mRNA levels of PGC-1α are increased. This suggests that cleaved PGAM5 might be involved with mitochondrial biogenesis by regulating transcription factors. It is known that PGAM5 interacts with β-catenin under mitochondrial stress in HEK293T and U2OS cells (17Bernkopf D.B. Jalal K. Bruckner M. Knaup K.X. Gentzel M. Schambony A. Behrens J. Pgam5 released from damaged mitochondria induces mitochondrial biogenesis via Wnt signaling.J. Cell Biol. 2018; 217: 1383-1394Crossref PubMed Scopus (42) Google Scholar). Therefore, we evaluated whether the cleaved PGAM5 binds with β-catenin during differentiation to activate β-catenin transcriptional activity using an immunoprecipitation assay. We found that β-catenin interacted with cleaved PGAM5 on day 4 (Fig. 4, E and F). Next, to evaluate if the stabilized β-catenin through interaction with PGAM5 translocates to the nucleus on day 4, we performed a subcellular fractionation assay of cells at different days of differentiation. Consistent with the increased levels of cleaved PGAM5, the nuclear β-catenin increased on days 2 and 4 (Fig. 4, G and H). To confirm these findings, we also performed immunohistochemistry staining for β-catenin and nuclei to visualize nuclear β-catenin (Fig. 4, I and J). We found an increase in nuclear β-catenin on day 4 as compared with control iPSCs. This further demonstrates increased β-catenin activity on day 4. Furthermore, we examined the β-catenin transcriptional activity during differentiation. hiPSCs were transfected with 8xTopFlash (TCF/LEF promoter) reporter luciferase and Renilla (control for transfection) and then differentiated for 2 or 4 days followed by luciferase assay. We found that β-catenin transcriptional activity on days 2 and 4 corresponded with an increase in nuclear β-catenin (Fig. 5A). These data suggest that mitophagy induces PGAM5 cleavage during differentiation and that cleaved PGAM5 dephosphorylates β-catenin, which promotes its translocation into the nucleus to express target genes. As we observed increased β-catenin activity on days 2 and 4, we performed a luciferase assay on cells with PGAM5 knockdown. We found that while transcriptional activity levels on day two did not change, there was a significant decrease in β-catenin activity on day 4 with PGAM5 knockdown (Fig. 5B), which coincided with PGAM5 cleavage. We next investigated whether β-catenin transcriptional activity promotes mitochondrial biogenesis. iPSC-ECs were treated with CHIR99021 (which activates Wnt/β-catenin signaling by inhibiting GSK3β) for 24 h and then evaluated expressional levels of the key regulator of mitochondrial biogenesis, the transcriptional coactivator PGC-1α. Upon Wnt/β-catenin activation, PGC-1α levels were significantly increased (Fig. 5C). To determine whether PGC-1α expression was dependent on PGAM5-induced β-catenin transcriptional activity, we examined the effect of PGAM5 depletion. hiPSCs were transduced with lentivirus encoding doxycycline inducible PGAM5 shRNA and differentiated for 7 days into iPSC-ECs. Following PGAM5 depletion, differentiated ECs had significantly lower PGC-1α when compared with control cells (Fig. 5, D and E). Taken together, our results suggest that PGAM5 is required for β-catenin transcriptional activity to induce PGC-1α expression, a known inducer for mitochondrial biogenesis during differentiation. The main objective of this study was to understand the role of mitophagy and mitochondrial biogenesis in mediating metabolic reprogramming during iPSC differentiation. We show that (1) mitophagy is increased early on during iPSC differentiation toward the endothelial lineage; (2) mitochondrial biogenesis increases after the initial phase of increased mitophagy during differentiation; (3) during iPSC differentiation, the phosphatase PGAM5 is cleaved during mitophagy and stabilizes β-catenin, thus signaling the initiation of compensatory mitochondrial biogenesis (Fig. 5F). Traditionally, mitophagy is considered to serve as a quality control mechanism, which eliminates damaged or dysfunctional mitochondria and is the setting of aging or stress (11Ng M.Y.W. Wai T. Simonsen A. Quality control of the mitochondrion.Dev. Cell. 2021; 56: 881-905Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 12Palikaras K. Lionaki E. Tavernarakis N. Mechanisms of mitophagy in cellular homeostasis, physiology and pathology.Nat. Cell Biol. 2018; 20: 1013-1022Crossref PubMed Scopus (476) Google Scholar, 25Lahiri V. Hawkins W.D. Klionsky D.J. Watch what you (self-) eat: Autophagic mechanisms that modulate metabolism.Cell Metab. 2019; 29: 803-826Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Inadequate mitophagy due to genetic mutations in mediators such as Mfn2 or Parkin (26Li Y.J. Cao Y.L. Feng J.X. Qi Y. Meng S. Yang J.F. Zhong Y.T. Kang S. Chen X. Lan L. Luo L. Yu B. Chen S. Chan D.C. Hu J. et al.Structural insights of human mitofusin-2 into mitochondrial fusion and CMT2A onset.Nat. Commun. 2019; 10: 4914Crossref PubMed Scop