TNF-Induced tic Cell Death

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Conserved metabolic energy production pathwaysgovern Eiger/TNF-induced nonapoptotic cell deathHiroshi Kandaa, Tatsushi Igakib,c, Hideyuki Okanoa, and Masayuki Miurad,e,1

aDepartment of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan; bDepartment of Cell Biology, Global Center of Excellence (G-COE),Kobe University Graduate School of Medicine, Hyogo 650-0017, Japan; cPrecursory Research for Embryonic Science and Technology (PRESTO), Japan Science

and Technology Agency, Saitama 332-0012, Japan;

d

Department of Genetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo113-0033, Japan; and eCore Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo 113-0033, Japan

Edited by Kathryn V. Anderson, SloanKettering Institute, New York, NY, and approved October 12, 2011 (received for review February 28, 2011)

Caspase-independent cell death is known to be important inphysiological and pathological conditions, but its molecular regu-

lation is not well-understood. Eiger is the sole fly ortholog of TNF.

The ectopic expression of Eiger in the developing eye primordium

caused JNK-dependent but caspase-independent cell death. Tounderstand the molecular basis of this Eiger-induced nonapoptotic

cell death, we performed a large-scale genetic screen in Drosophila

for suppressors of the Eiger-induced cell death phenotype. We

found that molecules that regulate metabolic energy production

are central to this form of cell death: it was dramatically sup-

pressed by decreased levels of molecules that regulate cytosolic

glycolysis, mitochondrial -oxidation of fatty acids, the tricarbox-ylic acid cycle, and the electron transport chain. Importantly, re-

ducing the expression of energy production-related genes did not

affect the cell death triggered by proapoptotic genes, such asreaper, hid, or debcl, indicating that the energy production-relatedgenes have a specific role in Eiger-induced nonapoptotic cell death.

We also found that energy production-related genes regulate the

Eiger-induced cell death downstream of JNK. In addition, Eiger

induced the production of reactive oxygen species in a manner

dependent on energy production-related genes. Furthermore, weshowed that this cell death machinery is involved in Eigers phys-

iological function, because decreasing the energy production-related

genes suppressed Eiger-dependent tumor suppression, an intrinsic

mechanism for removing tumorigenic mutant clones from epithe-

lia by inducing cell death. This result suggests a link between sen-

sitivity to cell death and metabolic activity in cancer.

necroptosis | chromosomal deficiency screen | tumor | scribble

Recent studies have revealed that nonapoptotic cell death isimportant in both physiological and pathological conditions;however, its molecular mechanism is still largely unknown. Ag-onistic ligands of death receptors, such as tumor necrosis factor-(TNF), FasL, and TNF-related apoptosis-inducing ligand(TRAIL), generally induce caspase-dependent cell death, thetypical form of apoptosis (1). Intriguingly, these ligands can alsoinduce nonapoptotic cell death when caspases are inhibited orcannot be activated efficiently (2, 3).

A surprising finding that implied the importance of non-

apoptotic cell death was that, except in the brain, mice mutantfor core regulators of apoptosis do not exhibit severe embryonictissue-sculpting defects such as interdigital tissue removal, aclassic example of physiological cell death (although bax/bak/

double mutant mice have some remaining interdigital tissue) (4).Interestingly, nonapoptotic cell death is observed in the inter-digital region ofapaf-1 mutant mice (5, 6). These results indicatethat nonapoptotic cell death may function physiologically toremove unnecessary tissue or as an alternative mechanism toremove cells when the apoptotic machinery is inhibited. Caspase-independent or nonapoptotic cell death has also been observedin oncogenic ras-induced cell death (7) and under other patho-logical conditions (8).

We and other groups previously reported that the Drosophilagenome encodes one pair of TNF/TNF receptor (TNFR) super-

family proteins: Eiger and its receptor, Wengen (912). The ec-topic expression of Eiger induces cell death through the activa-tion of c-Jun N-terminal kinase (JNK) signaling both in vivo (9,10) and in vitro (12). Intriguingly, Eiger-induced cell death seemsfar less sensitive to the baculovirus-derived pan-caspase inhibitorp35 (9, 10) than other apoptotic cell deaths (13). Thus, the Eiger-induced cell death signaling in Drosophila can provide a powerfulgenetic model system for studying the conserved mechanism ofTNF-induced, caspase-insensitive cell death signaling in vivo.

Although several downstream molecules that mediate Eiger-Wengen signaling have been identified (912, 1418), little is

known about the mechanism by which these molecules mediatenonapoptotic cell death. In mammals, the signaling cascade ofnonapoptotic cell death has been studied since the identificationof necroptosis, which is induced by TNF in the presence of cas-pase inhibitor (1921). However, no comprehensive genetic in-

vestigation to elucidate the mechanisms of nonapoptotic celldeath, programmed necrosis, or necroptosis in vivo has beenreported. Therefore, to examine the genetic control of non-apoptotic cell death, we chose a forward genetic screen for Eiger/TNF-induced cell death signaling in Drosophila.

In this study, we found that Eiger-induced nonapoptotic celldeath signaling requires molecules related to the production ofmetabolic energy downstream of activated JNK. The ectopicactivation of Eiger signaling resulted in the overproduction of

reactive oxygen species (ROS) downstream of JNK signaling. Wealso found that this cell death system is, at least in part, used asan intrinsic tumor suppression system in endogenous Eiger sig-naling to remove tumorigenic scrib mutant cells from epithelia.

Results

Eiger-Induced Cell Death Does Not Require the Canonical CaspaseActivation Pathway in Drosophila Developing Eyes. We previouslyshowed that the sole Drosophila TNF superfamily ligand, Eiger,induces massive cell death when it is ectopically expressed in thedeveloping eye primordium (eye imaginal disc). This cell deathrequires the activation of the Drosophila JNK, Basket (Bsk) (9,10). Intriguingly, although Eiger has been shown to activatecaspases (9, 10, 22, 23), the Eiger-induced eye phenotype wasonly slightly suppressed by coexpression of the pan-caspase in-

hibitor, p35 (Fig. 1 B and E) (9, 10), whereas p35 almost com-pletely suppressed the cell death phenotype triggered by theectopic expression of the proapoptotic gene reaper or head in- volution defective (hid) (Fig. 1 C, D, F, and G). This findingsuggested that the Eiger-induced cell death is only partly de-pendent on caspases. Therefore, we first sought to clarify the

Author contributions: H.K., T.I., and M.M. designed research; H.K. performed research;

H.K., T.I., and M.M. analyzed data; and H.K., T.I., H.O., and M.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

1073/pnas.1103242108/-/DCSupplemental.

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requirement for caspase activation in Eiger-induced cell death.Immunostaining for cleaved caspase 3 revealed that the over-expression of Eiger only slightly increased the caspase 3-likeprotease activity (Fig. 1 HJ). In addition, because p35 blocks theeffector caspases drICE and DCP1 but not the initiator caspaseDronc (24, 25), we evaluated Droncs involvement in the Eiger-induced cell death by overexpressing Eiger in a dronc-nullmutant background. However, no phenotypic suppression wasobserved under this condition (Fig. 1 KN), consistent witha previous observation that the overexpression of a dominantnegative form of Dronc does not affect the Eiger-induced eyephenotype (9). Thus, Eiger-induced cell death requires little,if any, activation of the canonical caspase pathway in the eye

imaginal disc.

Eighteen Deficiencies That Dramatically Suppress the Eiger-Induced

Eye Phenotype Identified by a Genome-Wide Dominant Modifier

Screen. To understand the molecular mechanism of the Eiger-induced nonapoptotic cell death in vivo, we conducted a ge-nome-wide screen using a series of chromosomal deficiency linesto identify dominant suppressors of Eiger-induced eye reduction(Materials and Methods). We screened more than 80% of thegenome and recovered 18 deficiency alleles, which we namedsuppressors of Eiger (SEs 118) (Fig. S1 and Table S1). Theresponsible regions for the suppression in these deficiency alleles

were narrowed down by using sets of small, partially overlappingdeficiencies, and the responsible genes were determined byscreening publicly available mutant lines. The Drosophila TNF

receptor, Wengen, was identified in the deficiency region of SE1,as described previously (11).

Knockdown of Genes Related to Energy Production Suppresses the

Eiger-Induced Small Eye Phenotype. We noticed that genes involvedor predicted to be involved in mitochondrial function were fre-quently found in the responsible regions of the SE lines ( TableS1). For instance, the Eiger-induced small eye phenotype wassignificantly suppressed by P-element insertions into or near the

loci ofcytochrome c-d (cyt.c-d; it encodes one of two isoforms ofDrosophila Cyt.c), Indy (a Krebs cycle intermediate transporterat the plasma membrane), Nmd (an ATPase), or CG18317 (amitochondrial carrier protein) (Table S1). In addition, the locusof Isocitrate dehydrogenase, which encodes a mitochondrial tri-carboxylic acid (TCA) cycle enzyme, was involved in a smalldeletion of Df(3L)66C-I65, which was recovered as the smalldeletion in SE8 (Table S1). Because the mitochondrion is themajor energy-producing organelle, we next evaluated the role ofmetabolic energy production-related genes in the regulation ofEiger-induced cell death. To examine the contribution of energyhomeostasis to Eiger-induced cell death, we knocked down en-ergy production-related genes in Eiger-expressing imaginal discs.Strikingly, the knockdown of genes involved in cytoplasmic gly-colysis, the mitochondrial TCA cycle, the -oxidation of fattyacids, or the electron transport chain was effective in suppressingthe Eiger-induced small eye phenotype (Fig. 2 A, F, and G).However, the knockdown of these genes had no effect on thecaspase-dependent cell death triggered by the overexpression ofReaper, Hid, or Debcl (Fig. 2 BD), indicating that regulation byenergy production-related molecules is specific for the Eiger-induced nonapoptotic cell death. These results indicated that theenergy production system is a crucial regulator of Eiger signaling.

Energy Production-Related Molecules Act Downstream of JNK

Signaling. Because Eiger-induced cell death is mediated by theJNK pathway (9, 10), we next examined the epistasis between theJNK pathway and the energy production-related genes that weidentified as suppressors of Eiger. The knockdown of carnitine-

palmitoyl transferase-I (CPTI; believed to be the rate-limiting stepof fatty acid oxidation), phosphoglycerate kinase (pgk; a trans-ferase in glycolysis), or cyt.c-d (electron transport chain protein)significantly inhibited cell death (Fig. 3 AE) without affectingJNK activation (Fig. 3 AE), suggesting that these moleculesmediate Eiger-induced cell death signaling downstream of JNK.

Eiger-Induced Cell Death Is Accompanied by the Production of ROS. Insome cell lines, such as the murine fibrosarcoma-derived L929,treatment with TNF induces caspase-independent cell deaththrough the production of ROS (26). We therefore examined ifEiger-induced cell death follows oxidative stress using flies thatexpress Glutathione S transferase D1-GFP (gstD-GFP), a ROS-inducible gstD promoter-GFP reporter (27, 28). When Eiger wasectopically expressed in the eye imaginal discs of these flies, the

cells showed an oxidative stress response, which was revealed bythe increase in gstD-GFP expression (Fig. 4 AC). The strengthof this response correlated with the strength of the adult eyephenotype (Fig. 4 AC and AC). Furthermore, dihydroethi-dium staining of these eye discs, in which Eiger was expressed insmall patches of the disc, indicated that the cells that ectopicallyexpressed Eiger produced superoxide (O2

) (Fig. S2). In addi-tion, the up-regulation of gstD-GFP reporter expression wasstrongly suppressed by reducing the gene dosage ofbsk (Fig. 4 C,C, D, D, and F). Furthermore, the ectopic expression of a JNKkinase kinase, dTAK1, also increased the gstD-GFP signal (Fig.S3). These results suggest that oxidative stress is induceddownstream of the activated JNK pathway.

We also found that the knockdown of energy production-re-lated genes such as cyt.c-d, GAPDH2, or aconitase significantly

FRT dronc A8

K L

GMR-GAL4, UAS-eiger

FRT droncA8

M N

Eiger Reaper

LacZ

p35

B C D

Hid

E F

wild-type

A

G

Cleaved

Caspase3

LacZ Eiger Hid

H I J

Fig. 1. Eiger-induced cell death is caspase-independent in Drosophila eyes.

(AG) Genetic interactions between the pan-caspase inhibitor p35 and Eiger,

Reaper, or Hid. Note that the coexpression of p35 with Eiger did not sig-

nificantly suppress the reduction of the adult eye size. ( HJ) Ectopic ex-

pression of Eiger did not induce a significant increase in caspase 3-like

activity. (KN) Loss of the Drosophila initiator caspase, dronc, did not sup-

press the Eiger-induced eye reduction. The dronc-null mutant allele is de-

scribed in ref. 49. In all images, anterior is to the left.

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attenuated the production of oxidative stress that was induced bythe ectopic expression of Eiger to a similar extent as the knock-down ofbsk or dTAK1 (Fig. 4G). In contrast, the overexpressionof Hid did not increase the gstD-GFP signal (Fig. 4 E, E, and F).These results, together with the findings shown in Fig. 3, suggestthat the energy production-related molecules regulate the pro-duction of ROS downstream of JNK in the Eiger-induced celldeath pathway.

Energy Production-Related Molecules Are Required for the Physio-

logical Eiger-Induced Death of Tumorigenic Mutant Cells. We nextassessed whether these molecules contribute to the cell death

triggered by endogenous Eiger. The imaginal tissue of mutantsfor evolutionarily conserved tumor suppressors, such as scribble(scrib) or discs large (dlg), overgrow and develop into tumors(29). However, when these tumorigenic mutant cells are sur-rounded by WT tissue, they do not overgrow but are, instead,eliminated from the tissue through EigerJNK-induced celldeath (30, 31). Thus, physiological Eiger signaling functions asan intrinsic tumor suppressor to eliminate tumorigenic mutantcells. We therefore asked if energy production-related moleculesregulate the endogenous Eiger signaling during the eliminationof tumorigenic scrib mutant clones. To this end, we reduced theexpression of CPTI (for fatty acid metabolism) or pgk (for gly-colysis) in scrib mutant clones by the Mosaic Analysis witha Repressible Cell Marker (MARCM) method (32). The scribmutant clones generated in the eye antennal discs were largely

eliminated from the tissue during the larval and pupal stages(Fig. 5 A, B, E, and F) (31). However, when CPTI or pgk wasknocked down, the area of the tumorigenic scrib mutant clones in

Indy

-oxidation

citrate

oxaloacetate

malate

f umarate succinate succinyl-CoA

-ketoglutarate

isocitratecis aconitate

acetyl-CoA

fatty acids

CPTI

cytosol

mitochondria

pyruvate

Glucose

glyceraldehyde 3-phosphate

1,3-bisphosphoglycerate

pyruvate

GAPDH

Pgk

3-phosphoglycerate

TCA cycle

Glycolysis pathway

walrus

F

dicarboxylates,

citrate

Idh

Mdh

Sdh

Acon Acon

PDH

G

NADH

FADH2

CoQI

IV

II

III

Cyt.c

TCA

cycle

2H2OO2 +4H+

mtACP1

Sdh

A

B

C

D

E

Fig. 2. Energy production-related proteins are specifically required to execute Eiger-induced cell death signaling. (AE) Light micrographs of transgenicflies.

The genetic background in AEwas w; UAS-eigerregg1/+; GMR-GAL4/+ (A), w; GMR-GAL4/+; GMR-rpr/+ (B), w; GMR- hid/+; GMR-GAL4/+ (C), w; GMR-GAL4/+;

UAS-debcl/+ (D), and w; GMR-GAL4/+; +/+ (E). Columns an indicate the responsible genes for each UAS-dsRNA line. Anterior is to the left. (Fand G) Schematic

diagram of glycolysis, -oxidation of fatty acids, and the TCA cycle ( F) and diagram of the electron transport chain (G). Responsible genes for RNAi lines that

suppressed the Eiger-induced phenotype are shown in red letters. Dashed arrows indicate that the corresponding genes have not been mapped to the

chromosome yet or RNAi flies are not publicly available. Complete genotypes and abbreviations are listed in SI Materials and Methods.

lacZ CPTI

GMR-GAL4, UAS-eiger, UAS-dsRNA

pgk cyt.c-d

p-JNK

A B C D

A B

AcridineOrange

C D

E

E

GMR-GAL4UAS-lacZ

Fig. 3. Energy production-related molecules act downstream of the JNK

pathway. Eye imaginal discs of wandering third instar larvae stained with an

anti-phospho-JNK antibody (AE) and acridine orange (AE). (AE) Eiger-

induced activation of JNK was not suppressed by the down-regulation of

energy production-related molecules. (AE) The extensive Eiger-induced

cell death posterior to the morphogenetic furrow was suppressed by the

down-regulation of energy production-related molecules. In all images,

anterior is to the left, and arrowheads indicate the morphogenetic furrow.

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the eye antennal discs increased significantly (Fig. 5 BD and I),although strong JNK activation was still observed in these cells(Fig. 5 EH and EH). The knockdown of CPTI or pgk in theWT cells did not increase the clone area or pupal lethality;rather, it decreased the clone area, presumably because of thereduction in energy production (Fig. 5I). Therefore, the in-creased area of the tumorigenic scrib mutant clone was not an

additive effect of the CPTI- or pgk-RNAi. Consistent with thisresult, a significantly greater number of animals carrying thesecells than control animals died as pupae (Fig. 5J). Together,these results indicated that energy production-related genes arecrucial for the endogenous Eiger-JNK-mediated cell death sig-naling that removes tumorigenic scrib cells from epithelia.

Discussion

Ectopic Expression of Eiger Induces Nonapoptotic Cell Death. In thispaper, we provide genetic evidence that metabolic energy pro-duction-related molecules are central to the Eiger/TNF-inducedcell death (Fig. 5K). We also provide evidence that this cell deathsystem is, at least in part, used in endogenous Eiger signaling toremove tumorigenic mutant cells from epithelia as an intrinsictumor suppression system.

It was recently revealed that at least some nonapoptotic celldeaths can be categorized as necroptosis, in which cells undergononapoptotic cell death under apoptosis-deficient conditions

when treated with agonistic ligands of death receptors, such asTNF, FasL, or TRAIL (33). The Eiger-induced cell deathshares features with necroptosis in that it is triggered by TNFfamily proteins, produces ROS, and is caspase-independent.Furthermore, the Drosophila homolog of a tumor suppressorprotein, Cylindromatosis, one of the essential regulators ofnecroptosis (34, 35), has been shown to regulate JNK activation

B

gstD-GFP,GMR-GAL4

UAS-lacZ

A

UAS-eigerst

C

UAS-eigerstbsk1

D

UAS-eigerwk

A B C D

+ +gstD-GFPGMR-GAL4 + +UAS-eiger

n= 31 17

dsRNA

st

**

numberofGFP+clusterpereyedisc

bsk

++st

18

acon

++st

29

dTAK1

++st

21

****G

++

33

cyt.c-d

st

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++st

26

GAPDH2

: p


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in Eiger-induced cell death signaling (16). However, despite thehigh conservation of most of the apoptotic machinery, blastsearch analysis has not identified Drosophila homologs of re-ceptor-interacting protein (RIP) 1 or RIP3, the essential kinasesfor inducing necroptosis (19, 20, 3639).

As we showed in Fig. S3, oxidative stress could be induceddownstream of the activated JNK pathway. However, we alsofound that the knockdown of energy production-related genessuch as cyt.c-d or GAPDH2 did not suppress the dTAK1- or

HepCA

-induced cell death phenotype (Fig. S4). This findingcould be caused by the overexpression of dTAK1- or HepCA-induced additional pathways, because we found that dTAK1 orHepCA overexpression induced JNK activation much morestrongly than Eiger overexpression (Fig. S5). Because dTAK1also functions downstream of Imd in the innate immune re-sponse, Imd-related signaling was another possible mechanismfor mediating Eiger signaling. Therefore, we examined the ge-netic interaction between Eiger-induced cell death and Imd orImd-related genes. However, we did not find any significantinteractions between them (Fig. S6). Therefore, it would be in-teresting to examine if other proteins can substitute for thefunctions of RIP1 or RIP3 in Eiger signaling or if fly TNF signal-ing uses other mechanisms to induce nonapoptotic cell death.

TNF-Induced Nonapoptotic Cell Death and Energy Production. It hasbeen reported that TNF-induced nonapoptotic cell death leadsto the RIP3-dependent activation of glycogen phosphorylase,

which is the rate-limiting enzyme in the degradation of glycogenand therefore, the key molecule for regulating energy production(39). In this context, ROS can be generated by the production ofexcess energy (39). This finding could explain why the down-regulation of energy production-related genes suppressed Eiger-induced cell death. However, we also observed that the amountof ATP in the eye antenna imaginal disc decreased when Eiger

was overexpressed, and this decrease in ATP was cancelled bythe knockdown of bsk, CPTI, pgk, or cyt.c-d (Fig. S7). Thisfinding suggests that the activation of energy production by Eigersignaling could also trigger another mechanism that decreases

the tissue ATP level. It is possible that the tissue loses ATPsimply because of the massive cell death caused by Eiger ex-pression. Alternatively, the work by Temkin et al. (40) reportedthat treatment with TNF and the caspase inhibitor zVAD notonly induces nonapoptotic cell death with ROS production butalso decreases ATP because of the inhibition of adenine nucle-otide translocase (ANT) by RIP1. A similar ANT-dependentinhibition mechanism could be involved in Eiger-induced celldeath. Because neither RIP1 nor RIP3 has yet been identified inDrosophila, the mechanism by which the total ATP is regulatedin Eiger-induced cell death remains to be elucidated.

Drosophila Model of Intrinsic Tumor Suppression. When scrib or dlgmutant cells are induced as clones in otherwise WT eye imaginaldiscs, most of these mutant cells are eliminated by EigerJNK-

dependent cell death during development (31, 41). This celldeath could involve a caspase-independent mechanism, becausethe elimination of mutant cells is not fully suppressed by theoverexpression of p35 compared with the blockage of JNK sig-naling (30, 31). Thus, the mode of cell death triggered in scribmutant clones is analogous to the mode of cell death triggered

by the overexpression of Eiger in imaginal discs. Our observa-tions suggest that the regulation of energy production could bea crucial determinant of the susceptibility of tumor cells to cy-totoxic stimuli.

Interestingly, tumor cells frequently produce ATP by glycolysisin the cytosol rather than in the mitochondria, which is knownas the Warburg effect (42, 43). Because mitochondrial energyproduction generates cytotoxic ROS, cancer cells might increasetheir resistance to cytotoxic stimuli by reducing mitochondrial

energy production (43). In this sense, mitochondrial energyproduction could act as a tumor suppressor. In fact, subunits of aTCA cycle enzyme, Succinate dehydrogenase (Sdh; SdhB, SdhC,and SdhD), are reported to be classical tumor suppressors inpheochromocytoma or paraganglioma (44). Furthermore, a spe-cific isoform of pyruvate kinase, which is involved in glycolysis, isnecessary for cellular metabolism to shift to aerobic glycolysisand the promotion of tumorigenesis (45). Similarly, the activityof pyruvate dehydrogenase (PDH), which links the glycolyticpathway to the TCA cycle by transforming pyruvate to acetyl-CoA, is suppressed in cancer cells, whereas the reactivation ofPDH induces cell death in a solid tumor cell line and xenografts(46). Interestingly, we found that the knockdown of DrosophilaPDH (which is encoded by CG7010) (47) strongly suppressed

Eiger-induced cell death (Fig. 2A, g). Furthermore, the down-regulation of genes involved in glycolysis and the -oxidation offatty acids significantly suppressed the elimination of scrib cellsfrom imaginal epithelia (Fig. 5). These observations suggest thatthe regulation of cellular energy production or even the source ofenergy could be critical for controlling the susceptibility of can-cer cells to cytotoxic stimuli such as TNF.

Materials and Methods

The deficiency kit flies were obtained from Bloomington Stock Center.

Transgenic flies for RNAi experiments were obtained from the Vienna Dro-

sophila RNAi Center and National Institute of Genetics. The UAS-eigerregg1

allele was described previously (9, 48). The gstD-GFPreporter fly (27, 28) was

used to detect the antioxidant response. Experiments were approved by the

committee on Living Modified Organisms of Keio University, Kobe Univer-

sity, and The University of Tokyo. Additional details are in SI Materialsand Methods.

ACKNOWLEDGMENTS. We thank Shu Kondo for sharing unpublished data,Hiroka Aonuma and Takahiro Chihara for helpful support, and Shu Kondoand Hirotaka Kanuka for discussions. We also thank Rieko Shimamura,Toshie Naoi, Naoko Tokushige, and Yuki Yamamoto-Goto for technicalsupport, Shizue Ohsawa for technical advice, Takeshi Yagi for kind supportand cooperation, and current and former H.O. and M.M. laboratorymembers for helpful discussions and comments. We thank Toshiro Aigaki,Sharad Kumar, Helena Richardson, Dirk Bohmann, Darren Williams, YasushiHiromi, Hermann Steller, Makoto Nakamura, the Bloomington Stock Center,the Drosophila Genetic Resource Center, the National Institute of Geneticsstock center (NIG-FLY), and the Vienna Drosophila RNAi center for fly stocks.This work was supported in part by grants from the Japanese Ministry ofEducation, Science, Sports, Culture, and Technology (MEXT; to H.K., T.I.,H.O., and M.M.), the Japan Society for the Promotion of Science (to H.K.),the Keio Gijuku Academic Development Funds (to H.K.), the Keio University

Grant-in-Aid for Encouragement of Young Medical Scientists (to H.K.), theStrategic Research Foundation Grant-Aided Project for Private Universitiesfrom MEXT (to H.K.), the Global Center of Excellence (G-COE) program forGlobal Center for Education and Research in Integrative Membrane Biology(to T.I.), the International Human Frontier Science Program (to T.I.), theJapan Science and Technology Agency (to T.I. and M.M.), and grant-in-aidfor the G-COE program from MEXT to Keio University (to H.K. and H.O.).

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Supporting Information

Kanda et al. 10.1073/pnas.1103242108

SI Materials and Methods

Genetic Screen. Fly culture and crosses were carried out at 25 Cunless otherwise specified. Canton-S was used as the WT strain.

A series of Bloomington defi

ciency kit lines were crossed withUAS-eigerregg1 ; GMR-GAL4/TM3 transgenic flies, and the F1progenies were examined for suppression of the small eye phe-notype. If a chromosomal deficiency region contained a genethat functions downstream of Eiger, the Eiger-induced small eyephenotype should be suppressed. More than 80% of the Dro-sophila genome was analyzed in the primary screen. The re-sponsible regions for phenotype suppression were narroweddown by using small, overlapping deficiencies. The candidatesfor responsible genes were then screened by examining publiclyavailable mutants. To obtain images of the adult eye, flies werefrozen at 80 C for 5 min, and photographs were taken andprocessed with an Olympus SZX16 camera with Dynamic EyeREAL software (Mitani).

Fly Food. To avoid variations in energy supply from theflyfood, weusedfreshly cookedfly food according to thefollowing recipe inallof theexperiments:400 g dry yeast (OrientalDry Yeast), 400g cornflour (Nippn), and 80 g agar (Kishida Chemical) were mixed andboiled well in 9 L water. Subsequently, 1,000 g glucose (NakalaiTesque) weredissolved in 1 L hotwater, and addedto themixture;30 mL propionic acid (Nakalai Tesque) and 50 mL 10% butyl p-hydroxybenzoicacidin70%ethanolwereaddedbeforealiquotting.

Histology. Eye discs from third instar larvae were stained bystandard immunohistochemical procedures using a rabbit anti-phospho-JNK monoclonal antibody (1:100; Cell Signaling) orrabbit anti-cleaved caspase 3 antibody (1:100; Cell Signaling).The secondary antibodies were 555 Alexa anti-mouse IgG(1:1,000; Molecular Probes) and 555 Alexa anti-rabbit IgG

(1:1,000; Molecular Probes). The stained samples were mountedwith antifade reagent (Slow Fade Gold; Invitrogen). For acri-dine orange staining, dissected eye discs were incubated with 1.6M acridine orange (Sigma) solution for 2 min (1). After a briefrinse with PBS, the samples were analyzed by fluorescencemicroscopy.

Dihydroethidium Staining. Eye discs of third instar larvae weredissected in PBS and then stained in 5 M dihydroethidium(Invitrogen)/PBS solution for 5 min at room temperature. Thediscs were then briefly rinsed with PBS two times and washed

with PBS for 5 min. The fluorescence was analyzed undera fluorescence microscope.

Monitoring of Cellular Reactive Oxygen Species Production by a gstD-

GFP Reporter. The eye discs of transgenicflies containing a gstD-GFPreporter construct were dissected and stained using an anti-GFPantibody (1:100; MBL). The secondary antibody was 488 Alexa anti-rabbit IgG (1:1,000; Molecular Probes). The samples were mounted

with VECTASHIELD mounting medium (Vector Laboratories).The number of GFP-positive clusters was counted under a fluo-rescence microscope.

ATP Assay. The amount of ATP in the eye antenna imaginal discswas analyzed using an ATP determination kit (A22066; MolecularProbes). In brief, the discs were dissected in PBS, and each disc

was separately placed into 1 reporter lysis buffer (Promega).The samples were immediately heat-shocked at 95 C to in-

activate ATPase and then quickly frozen. The tissue lysates werecentrifuged at 15,300 g for 5 min, and the supernatant wassubjected to the ATP assay. The luminofluoresence was mea-sured by a Lumat LB9507 (Berthold).

Detailed Genotypes of the Animals Used in Figures.

Fig. 1A: WT (Canton-S)Fig. 1B: w; UAS-eigerregg1/UAS-lacZ; GMR-GAL4/+Fig. 1C: w; GMR-GAL4/UAS-lacZ; GMR-rpr/+Fig. 1D: w; GMR-hid/UAS-lacZ; GMR-GAL4/+Fig. 1E: w; UAS-eigerregg1/+; GMR-GAL4/UAS-p35Fig. 1F: w; GMR-GAL4/+; GMR-rpr/UAS-p35Fig. 1G: w; GMR-hid/+; GMR-GAL4/UAS-p35Fig. 1H: w; UAS-lacZ/GMR-GAL4Fig. 1I: w; UAS-eigerregg1/GMR-GAL4Fig. 1J: w; GMR-hid/+Fig. 1K: yw eyFLP/+; GMR-hid l(3)* FRT2A/FRT2AFig. 1L: yw eyFLP/+; GMR-hid l(3)* FRT2A/droncA8 FRT2A

Fig. 1M: yw eyFLP/+; GMR-GAL4, UAS-eiger8/+; GMR-hid,l(3)* FRT2A/FRT2A

Fig. 1N: yw eyFLP/+; GMR-GAL4, UAS-eiger8/+; GMR-hid, l(3)* FRT2A/ droncA8 FRT2A

The transgenic RNAi flies used in the experiments in col-umns an of Fig. 2 were UAS-lacZ-inverted repeat (IR; a), UAS-CPTI-IRVDRC4046 (b), UAS- cyt.c-d-IRVDRC17129 (c), UAS-wal-rus-IRVDRC44378 (d), UAS- pgk-IRVDRC33798 (e), UAS-SdhA-IRVDRC110440 (f), UAS- PDH-IRVDRC40410 (g), UAS-GAPDH1-IRVDRC31631 (h), UAS-GAPDH2-IRVDRC23645 (i), UAS-acon-IRVDRC11767 (j), UAS-Idh-IRVDRC42915 (k), UAS-Indy-IRVDRC9981

(l), UAS-mtacp1-IRVDRC43503 (m), and UAS-Mdh-IRVDRC27535 (n).

Fig. 3 A and A

: w; UAS-lacZ/+; GMR-GAL4/+Fig. 3 B and B : w; UAS-eigerregg1 /UAS-lacZ-IR; GMR-GAL4/+Fig. 3 C and C : w; UAS-eigerregg1/+; GMR-GAL4/UAS-CPTI-

IRVDRC4046

Fig. 3 D and D : w; UAS-eigerregg1/+; GMR-GAL4/UAS-pgk-IRVDRC33798

Fig. 3 E and E : w; UAS-eigerregg1/UAS-cyt.c-d-IRVDRC17129;GMR-GAL4/+

Fig. 4 A and A : w; gstD-GFP, GMR-GAL4/UAS-lacZFig. 4 B and B : w; gstD-GFP, GMR-GAL4/UAS-eigerwk

Fig. 4 C and C : w; gstD-GFP, GMR-GAL4/UAS-eigerst

Fig. 4 D and D : w; gstD-GFP, GMR-GAL4/UAS-eigerst , bsk1

Fig. 4 E and E : w; GMR-hid/gstD-GFPFig. 5 A, E, and E : yw, eyFLP1/+; act >y+ > Gal4, UAS-GFP/+;

FRT82B, Tub-Gal80/FRT82B

Fig. 5 B, F, and F : yw, eyFLP1/+; act > y+> Gal4, UAS-GFP/

UAS-lacZ-IR; FRT82B, Tub-Gal80/FRT82B, scrib1

Fig. 5 C, G, and G : yw, eyFLP1/+; act > y+ > Gal4, UAS-GFP/+; FRT82B, Tub-Gal80/UAS-CPTI-IRVDRC4046 FRT82B,scrib1

Fig. 5 D, H, and H : yw, eyFLP1/+; act > y+ > Gal4, UAS-GFP/+; FRT82B, Tub-Gal80/UAS-pgk-IRVDRC33798 FRT82B,scrib1

The genotypes of the control animals in which CPTI- or pgk-RNAi was induced in the WT clone in Fig. 5 I and J were yw,eyFLP1/+; act > y+ > Gal4, UAS-GFP/+; FRT82B, Tub-Gal80/UAS-CPTI-IRVDRC4046 FRT82B (CPTI), and yw, eyFLP1/+; act >

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y+ > Gal4, UAS-GFP/+; FRT82B, Tub-Gal80/UAS-pgk-IRVDRC33798 FRT82B (pgk), respectively.

Abbreviations in Fig. 2 are as follows: acon, aconitase; CPTI,carnitine palmitoyltransferase I; cyt.c-d, cytochrome c-distal; Idh,

Isocitrate dehydrogenase; Indy, Im not dead yet; IR, invertedrepeat; Mdh, malate dehydrogenase; mtacp1, mitochondrial acylcarrier protein 1; Pgk, phosphoglycerate kinase; Sdh, succinatedehydrogenase.

1. Miron M, et al. (2001) The translational inhibitor 4E-BP is an effector of PI(3)K/Akt

signalling and cell growth in Drosophila. Nat Cell Biol 3:596e601.

Fig. S1. Suppression of the Eiger-induced small eye phenotype by suppressors of Eiger (SE) alleles identi fied in the screen. Light micrographs of adult fly eyes.

For all flies except WT (Canton-S) and GMR-GAL4, UAS-eiger (UAS-eigerregg1/+; GMR-GAL4/ +), the genotype was UAS-eigerregg1/+; GMR-GAL4/+ on the het-

erozygous background for the deficiencies mentioned in the panels. The name, cytology of the deficiency region, and Bloomington stock number for each line

are summarized in Table S1.

Fig. S2. Activation of Eiger signaling induces the production of superoxide. Eye discs were stained with dihydroethidium (DHE), which is an indicator of

superoxide (O2). DHE-positive cells were detected in the clones in which Eiger was overexpressed. Genotypes were yw, eyFLP1/+; act> y+ > Gal4, UAS-GFP/

UAS-lacZ; FRT82B, Tub-Gal80/FRT82B (Upper) and yw, eyFLP1/+; act> y+ > Gal4, UAS-GFP/UAS-eiger12 ; FRT82B, Tub-Gal80/FRT82B (Lower), respectively. Ar-

rowheads indicate the morphogenetic furrow. Anterior is to the top.



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Fig. S3. Ectopic expression of dTAK1 increases the gstD-GFP signal. Eye discs were dissected, and the gstD-GFP signal was examined. The activation of JNK

signaling was detected by the immunostaining of anti-phospho-JNK. Genotypes were as follows: w; GMR-GAL4, gstD-GFP/UAS-lacZ(AC) and w; GMR-GAL4,

gstD-GFP/UAS-dTAK1 (DF). Anterior is to the left. Arrowheads indicate the morphogenetic furrow. Yellow arrow indicates the gstD-GFP signal, which was

induced by the overexpression of dTAK1.

Fig. S4. Down-regulation of cyt.c-d or GAPDH2 does not suppress the dTAK1- or HepCA-induced eye phenotype. Light micrographs of transgenic flies.

Genotypes were as follows: w; GMR-GAL4, UAS-lacZ-IR/+; UAS-dTAK1/+ (A), w; GMR-GAL4, UAS-cyt.c-d-IRVDRC17129/+; UAS-dTAK1/+ (B), w; GMR-GAL4, UAS-

GAPDH2-IR VDRC23645/+; UAS-dTAK1/+ (C), w; GMR-GAL4, UAS-lacZ-IR/+; UAS-hepCA/+ (D), w; GMR-GAL4, UAS-cyt.c-d-IRVDRC17129/+; UAS-hepCA /+ (E), and w;

GMR-GAL4, UAS-GAPDH2-IR VDRC23645/+; UAS-hepCA/+ (F). Anterior is to the left. Animals were raised at 18 C to avoid lethality.

Fig. S5. Overexpression of dTAK1 or HepCA strongly activates JNK. Eye discs were dissected, and the activation of JNK signaling was detected by the im-

munostaining of anti-phospho-JNK. Genotypes were as follows: w; GMR-GAL4/UAS-lacZ(A), w; GMR-GAL4/ UAS-eigerregg1 (B), w; GMR-GAL4/ UAS-dTAK1 (C),

and w; GMR-GAL4/ UAS-hepCA (D). Anterior is to the left. Arrowheads indicate the morphogenetic furrow.



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Fig. S6. Lackof a geneticinteraction betweenEiger-inducedcelldeath andImd-relatedgenes. Genotypeswere as follows:w; UAS-eigerregg1/UAS-lacZ-IR;GMR-GAL4/+

(A), w; UAS-eigerregg1/+; GMR-GAL4/relE20 (B), w; UAS-eigerregg1/UAS-Imd-IR5516R-1; GMR-GAL4/+(C),and w; UAS-eigerregg1/UAS-Dredd-IR7486R-2; GMR-GAL4/+(D).

Fig. S7. Activation of Eigersignalingleads to a reductionin ATP.The eyeantennaimaginaldiscsof third instar larvae weredissected andsubjected to an ATPassay. The

amount of ATP in individual eye antenna imaginal discs was analyzedseparately. Genotypes: UAS-lacZ/+; GMR-GAL4/+(GMR> lacZ), UAS-eigerregg1/+; GMR-GAL4/+(),

UAS-eigerregg1/UAS-lacZ-IR; GMR-GAL4/+ (lacZ), UAS-eigerregg1/+; GMR-GAL4/UAS-bsk-IRVDRC104569 (bsk), UAS-eigerregg1/+; GMR-GAL4/UAS-pgk-IRVDRC33798 (pgk), UAS-

eigerregg1/+; GMR-GAL4/UAS-CPTI-IRVDRC4046 (CPTI), and UAS-eigerregg1/UAS-cyt.c-d-IRVDRC17129; GMR-GAL4/+ (cyt.c-d). All data are presented as the mean SEM

(black bar) of the number of eye discs indicated by n. Asterisks indicate statistical significance determined by Students ttest. **P< 0.005; **P< 0.001.

Table S1. Eighteen deficiency lines that suppressed the Eiger-induced eye reduction

SE BL Allele Small df Responsible region Allele Candidate gene (Predicted) molecular function

1 3,070 Df(1)E128 None 17C; 18A see ref. 1 wgn TNF receptor

2 967 Df(1)C246 None 11D-E; 12A01-02 hep1 hep JNKK

3 1,682 Df(2R)or-BR6 2 59F3; 60A8-16 CG11299BG01215 CG11299

4 2,471 Df(2R)M60E None 60E02-03; 60E11-12 zip1 zip Cytoskeletal protein binding

5 2,583 Df(2L)cact-255rv64 1 36A8-9; 36D bln1 cyt-c-d Electron transport6 3,133 Df(2L)dp-79b 1 22A6-22B9 CG18317BG02028 CG18317 Mitochondrial carrier

7 6,299 Df(2L)BSC5 None 26B1-2; 26D1-2

8 1,541 Df(3L)66C-G28 1 66C07-10; 66C7-10 Idh Isocitrate dehydrogenase

Nmtj1C7 Nmt Glycylpeptide

N-tetradecanoyltransferase

9 1,962 Df(3R)p-XT103 4 85A04-05; 85A06-11

10 1,990 Df(3R)Tpl10 None 83C01-02; 84B02 Scr17 Scr Transcription factor

ftzUal2rv3 ftz Transcription factor

zen2 zen Transcription factor

11 2,611 Df(3L)vin5 4 68C08-11; 68D6 CG5946BG01087 CG5946 Cytochrome-b5 reductase

12 2,990 Df(3L)Cat 1 075B08; 075F01 W05014 hid Cell death induction

I(3)j14E7j14E7 Indy Transporter

CG6896BG01140 CG6896 Phosphatase

13 3,011 Df(3R)Cha7 4 90F01-04; 91A1-2

14 3,547 Df(3R)L127 None 99B05-06; 99E04-F01

15 5,411 Df(3L)Aprt-32 1 62B07; 62B12

16 4,431 Df(3R)DG2 4 90F01-04; 91A01-02

17 1,045 Df(2L)Mdh None 30D-F; 31F bsk1, bsk2 bsk JNK

nmdk10909 Nmd (no mitochondrial

derivative)

ATPase, carrier

18 950 Df(1)RA2 None 7D18; 8A4

The suppressor of Eiger (SE) number, Bloomington stock number (BL), deficiency name, number of overlapping small deficiencies, cytology of the de-

termined responsible region, allele name that dominantly suppressed the Eiger-induced eye phenotype, predicted responsible gene of the allele, and

molecular function of the putative responsible gene product are listed. Energy metabolism-related molecules are highlighted in red letters.

1. Kanda H, Igaki T, Kanuka H, Yagi T, Miura M (2002) Wengen, a member of the Drosophila tumor necrosis factor receptor superfamily, is required for Eiger signaling. J Biol Chem 277:

28372e28375.


TNF-Induced tic Cell Death

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