PEA15) Form a Molecular Switch Governing Cellular Fate Depending on the Metabolic State
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Mitochondrial hexokinase II (HKII) and phosphoprotein enriched in astrocytes (PEA15) form a molecular switch governing cellular fate depending on the metabolic state Philipp Mergenthalera,b,c,d,1,AnjaKahla,c, Anne Kamitza,c, Vincent van Laake, Katharina Stohlmanna,c, Susanne Thomsena,c, Heiko Klawittera,c, Ingo Przesdzinga,c, Lars Neeba,b,c, Dorette Freyera,c, Josef Prillerf, Tony J. Collinsg,h, Dirk Megowa,c, Ulrich Dirnagla,b,c, David W. Andrewsg,h,1, and Andreas Meisela,b,c,d aDepartment of Experimental Neurology, bDepartment of Neurology, cCenter for Stroke Research Berlin, dNeuroCure Clinical Research Center, eDepartment of Internal Medicine, Pulmonary Medicine, and Infectious Diseases, and fNeuropsychiatry and Laboratory of Molecular Psychiatry, Charité Universitätsmedizin Berlin, 10117 Berlin, Germany; and gDepartment of Biochemistry and Biomedical Sciences, hMcMaster Biophotonics Facility, McMaster University, Hamilton, ON, Canada L8N 3Z5 Edited by Douglas R. Green, St. Jude Children’s Research Hospital, Memphis, TN, and accepted by the Editorial Board December 14, 2011 (received for review May 24, 2011) The metabolic state of a cell is a key determinant in the decision to live elusive. We therefore aimed to investigate the complex functional and proliferate or to die. Consequently, balanced energy metabolism roles of mitochondrial HKs in the adaptive response to different and the regulation of apoptosis are critical for the development and states of metabolic deprivation during hypoxia and hypoglycemia. maintenance of differentiated organisms. Hypoxia occurs physiolog- ically during development or exercise and pathologically in vascular Results disease, tumorigenesis, and inflammation, interfering with homeo- HIF-1–Dependent Activation of HKII in Primary Neurons and Hypoxia static metabolism. Here, we show that the hypoxia-inducible factor Tolerance. First, we assessed whether mitochondrial hexokinases (HIF)-1–regulated glycolytic enzyme hexokinase II (HKII) acts as a mo- responded to a hypoxia-mimicking stimulus that activates HIF-1 lecular switch that determines cellular fate by regulating both cytopro- transcriptional activity by interfering with degradation of HIF-1α tection and induction of apoptosis based on the metabolic state. We (19). We treated cultured primary rat brain cortical neurons with CELL BIOLOGY provide evidence for a direct molecular interactor of HKII and show the iron chelator deferoxamine (DFO), thereby mimicking hypoxia that, together with phosphoprotein enriched in astrocytes (PEA15), (19). DFO treatment resulted in marked protection from neuronal HKII inhibits apoptosis after hypoxia. In contrast, HKII accelerates ap- cell death after oxygen-glucose deprivation (OGD), an established model of cerebral ischemia (Fig. 1). At specified time points (Fig. optosis in the absence of PEA15 and under glucose deprivation. HKII 1A), we extracted RNA or protein from sister cultures to assess both protects cells from death during hypoxia and functions as a sensor changes in gene expression for both HKI (Fig. 1B)andHKII(Fig. of glucose availability during normoxia, inducing apoptosis in response 1C). HKI is the predominating isoenzyme in the brain and HKII is to glucose depletion. Thus, HKII-mediated apoptosis may represent an typically expressed in insulin-sensitive tissues or in malignant tumors evolutionarily conserved altruistic mechanism to eliminate cells during (20). However, messenger RNA (mRNA) expression of HKII was metabolic stress to the advantage of a multicellular organism. increased at 12 h after DFO treatment, whereas HKI expression did not change over 48 h. Immunoblotting of proteins from these cul- cell death | endogenous tolerance | fluorescence lifetime imaging tures revealed a concordant increase in HKII protein content (Fig. microscopy-FRET | mitochondria | preconditioning 1D). Using isoenzyme-specific electrophoretic zymography, we further demonstrated a specific increase in HKII activity (factor of alanced energy metabolism and regulation of apoptosis are of average increase = 2.28 ± 0.54), but not HKI activity in response to Bvital importance to all organisms (1, 2). Therefore, energy DFO treatment (Fig. 1 E and F). At 48 h after DFO treatment, metabolism and regulation of apoptosis are interdependent (3). In neurons were protected from cell death, as indicated by decreased cells, metabolism and apoptosis converge at mitochondria, thereby lactate dehydrogenase (LDH) release 24 h after OGD (Fig. 1G). integrating pathways responsible for endogenous tolerance against We therefore hypothesized that up-regulation of HKII significantly substrate deprivation (4). All differentiated multicellular organ- contributes to hypoxia tolerance mediated by activation of HIF-1. isms have evolved strategies to promote survival when deprived of metabolic substrates, such as during hypoxia (5). Therefore, elu- Overexpression of HKII Protects Primary Neurons from Hypoxic Cell cidating the underlying molecular mechanisms by which metabo- Death. To study the functional relevance of the increase in HKII lism and apoptosis are coregulated may lead to novel therapeutic expression and activity, we investigated the effect of overexpressed strategies for both acute and chronic diseases. HKII in comparison with BclXL under hypoxic conditions. We The transcription factor hypoxia-inducible factor (HIF)-1 is a key chose BclXL as a positive control because of its strong antiapoptotic regulator in the adaptation to hypoxia and the resultant energy properties (21) and its role in preconditioning-induced neuro- depletion, orchestrating the cellular response to hypoxic conditions protection (22). Transient transfection typically targets only a sub- (5–7). Induction of HIF-1 leads to the transcriptional regulation of set of cells (∼30% of neurons in a culture). Therefore, we visualized a multitude of genes, ultimately resulting in a hypoxia-tolerant state transfected cells using fluorescence after transfection of plasmids of the cell (7). HIF-1 also links hypoxia and glycolysis (8) via complex and incompletely understood mechanisms. HIF-1 adapts cellular metabolism to hypoxic conditions during development (7) Author contributions: P.M., D.W.A., and A.M. designed research; P.M., A. Kahl, A. Kamitz, or exercise (9) and thereby prevents death of tumor cells and pri- V.v.L., K.S., S.T., H.K., I.P., L.N., D.F., J.P., and D.M. performed research; P.M., T.J.C., D.M., mary cells under various conditions of disease (5–7, 10). In addition, U.D., D.W.A., and A.M. analyzed data; and P.M., D.W.A., and A.M. wrote the paper. HIF-1 controls innate immunity by regulating glycolysis in cells of The authors declare no conflict of interest. the immune system (11, 12). Finally, by controlling the expression This article is a PNAS Direct Submission. D.R.G. is a guest editor invited by the Editorial of members of the glycolytic cascade, including hexokinase II Board. (HKII) (7, 8), HIF-1 contributes to a proliferative metabolism (13). 1To whom correspondence may be addressed. E-mail: [email protected] or Mitochondrial glycolytic hexokinase isoenzymes (HKI, HKII, or [email protected]. also HKIV) may mediate cytoprotection under various conditions This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (14–18). However, the molecular mechanisms have remained 1073/pnas.1108225109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1108225109 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 catalytic activity is required for HKII to sense the metabolic state of A 48h Day 8 protein, Day 0 the cell, thereby protecting cells from hypoxic death but inducing preconditioning HK activity, 72h plate neurons 150 mM DFO / no DFO OGD LDH cell death upon glucose starvation. HKII Regulates Apoptotic Signaling After Metabolic Disturbance but 0 6h 12h 18h 24h 48h Not After Genotoxic Injury. Next, we investigated whether HKII mRNA extraction would protect cells from apoptosis in general or only when cells B C were stressed by metabolic disturbances (e.g., hypoxia). Trans- 12 12 control control fected HeLa cells were submitted to hypoxic conditions for 6 h 10 150μM DFO 10 150μM DFO A B 8 N=7 8 N=7 (Fig. 3 ) with 24 h of reoxygenation or for 21 h (Fig. 3 ). Al- 6 6 ternatively, apoptosis was induced by treatment with 100 nM 4 4 actinomycin D (Fig. 3C)or75μM etoposide (Fig. 3D). Cell death 2 2 was analyzed in transfected cells using flow cytometry. Although HKI - fold induction 0 HKII - fold induction 0 0h 6h 12h 18h 24h 48h 0h 6h 12h 18h 24h 48h HKII rescued cells from apoptosis after hypoxia irrespective of severity (Fig. 3 A and B), it did not rescue cells after actinomycin D E F G D or etoposide treatment (Fig. 3 C and D). BclXL, used as positive 20 * 20 * fi N=7 N=7 control, mediated signi cant protection from cell death in both - DFO + DFO 15 15 models. These data demonstrate that HKII regulates apoptosis < application - DFO + DFO after metabolic disturbances but not after genotoxic injury. 10 HKII 10 5 Identification of Phosphoprotein Enriched in Astrocytes as Direct Actin HKI 5 % HK II activity of total HK activity neuronal cell death Interactor of HKII. To investigate the molecular mechanism that 0 [%LDH (OGD - control)] HKII 0 governs HKII-dependent regulation of apoptosis, we screened a mouse brain cDNA library for putative interactors of HKII using no DFO no DFO a membrane-based split-ubiquitin yeast two-hybrid system (Fig. 150 μM DFO 150 μM DFO 4A). Using this approach, we identified phosphoprotein enriched – in astrocytes (PEA15) as a candidate for interaction with HKII. To Fig. 1. Hypoxia tolerance mediated by HIF-1 dependent activation of HKII fi in primary neurons. (A) Diagram of experimental paradigm. Analysis of HKI investigate the regulation of PEA15, we rst measured its ex- (B, E, and F) and HKII (C–F) expression in response to DFO treatment. (C) HKII pression after hypoxia-mimicking activation of HIF-1. In contrast expression was induced 12 h after hypoxia-mimicking treatment. After 48 h, to HKII (Fig. 1), PEA15 mRNA was not regulated under these (D) HKII protein and (E and F) enzyme activity were also increased.