Misexpression of the Eyes Absent Family Triggers the Apoptotic ...

Eya Overexpression Induces Apoptosis

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Misexpression of the Eyes Absent Family Triggers the Apoptotic Program*

S. Wesley Clark,‡§ Brian E. Fee,‡ and John L. Cleveland‡§¶

From the Department of Biochemistry,‡ St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105; and the Department of Molecular Sciences,§ The University of Tennessee Health Science Center, Memphis, TN 38163.

* This research was supported by grants from the NCI and NIDDK, National Institutes of Health (to J. L. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ To whom all correspondence should be addressed: Department of Biochemistry, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Tel.: (901) 495-2398; Fax: (901) 525-8025; E-mail: [email protected].

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on November 7, 2001 as Manuscript M108410200 by guest on M

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Genetic studies in Drosophila and mice have shown that eyes absent (eya1) is an

important and conserved transcriptional regulator of development. Along with eyeless/Pax6,

sine oculis, and dachshund, eya genes function as master regulators in eye development and

can induce ectopic eye formation. Furthermore, loss-of-function mutants of these genes in the

fly causes partial or complete loss of the compound eye, and this is associated with

inappropriate apoptosis. Conversely, ectopic eyeless expression in the context of eyes absent

or sine oculis mutations results in apoptosis, suggesting that the proper ratio of these factors

regulates apoptosis. Here we report that enforced expression of fly eyes absent (eya), or of one

of its mammalian homologs, Eya2, triggers rapid apoptosis in Interleukin-3-dependent 32D.3

murine myeloid cells, which express Eya family members but not Pax6. Eya-induced cell

death overrides survival factors and has many features typical of apoptosis, including plasma

and mitochondrial membrane changes and caspase activation. Eya-induced apoptosis is

blocked by Bcl-2 overexpression, but not by the broad-spectrum caspase inhibitor z-

VAD.fmk, suggesting that mitochondria are a major target in Eya-induced apoptosis. These

results support the concept that inappropriate changes in the steady state levels of Eya

proteins may trigger programmed cell deaths during development.

1The abbreviations used are: eya, eyes absent; IL-3, interleukin-3; RT-PCR, reverse transcriptase polymerase chain reaction; PARP, Poly-ADP ribose polymerase; EST, expressed sequence tag; RACE, Rapid Amplification of cDNA ends; PI, propidium iodide; FACS, fluorescence activated cell sorter; FCS, fetal calf serum; NTP, nucleoside triphosphate; DiOC6, 3,3-dihexyloxacarbocyanine iodide; TMRM, tetramethylrhodamine ethyl ester; PBS, phosphate buffered saline; FITC, fluoroscein isothiocyanate; DMSO, dimethylsulfoxide; TBS, Tris-buffered saline.

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The development of the compound eye in the Drosophila eye-antennal imaginal disc

involves a complex sequence of events that include cell proliferation, differentiation, migration and

death (1, 2). During larval stages, a small set of progenitor cells proliferate to form the eye disc.

This is followed by a wave of differentiation that occurs when a morphogenetic furrow sweeps

across the eye field in a posterior to anterior fashion. In the wake of this furrow, cells differentiate

into cell clusters that eventually form the ommatidia of the eye. By contrast, cells continue to

proliferate ahead of the furrow, and as the furrow moves anteriorly their cell cycle becomes

synchronized (3).

Mutagenesis studies have established that key regulators in Drosophila eye development

include the transcription factors eyeless, sine oculis, dachshund, and eyes absent (eya). All are

highly conserved throughout evolution and play essential roles in eye formation (1,4-7). For

example, ectopic expression of eyeless, or its murine homolog Pax6, is sufficient to promote ectopic

eye development (8). Similarly, ectopic expression of eya along with either sine oculis or

dachshund is capable of inducing ectopic eyes (9-11). Further, there appears to be a hierarchical

regulation, such that eyeless regulates the expression of eya, sine oculis and dachsund (9, 12, 13).

In addition, feedback loops exist such that once eyeless induces the expression of eya, sine oculis

and dachsund, these factors are then capable of regulating one another's expression, ensuring the

proper implementation of the eye development program (12). Once the program is initiated other

downstream eye targets are activated, including those involved in photoreceptor development,

which involves the induction of dectapentaplegic and the repression of wingless (14). Finally, this

network is even more complex in vertebrates, which have multiple orthologs of each fly gene. For

example, four murine homologs of eyes absent have been identified (Eya1, Eya2, Eya3, and Eya4)

(15-18).


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A common denominator of the eyeless, sine oculis, dachshund, and eya mutations is either

partial or complete loss of the compound eye (1,4-7), and this is associated with the deaths of

progenitor cells located anterior to the morphogenetic furrow (1,4-7). In addition, when eyeless is

ectopically overexpressed in the wing disc of eya or sine oculis mutants, it promotes ectopic cell

death rather than eye development (12). Similarly, dachshund overexpression in either the

antennal, leg or wing disc also leads to inappropriate cell death (13).

The precise role these developmental regulators perform in controlling programmed cell

deaths is unresolved. It has been suggested that eya may inhibit cell death either directly or

indirectly as a default pathway that occurs following inappropriate proliferation or differentiation

(1). Immortal interleukin-3 (IL-3)-dependent 32D.3 murine myeloid cells provide an excellent

system to evaluate apoptotic regulators as, similar to primary hematopoietic progenitors, they

continuously require hemopoietins for their survival, and in vivo this fail-safe mechanism strictly

regulates hematopoietic cell numbers (19-23). Expression analyses demonstrated that the Eya

family genes Eya1-3 were expressed in these cells, but that they failed to express Pax6, suggesting

that this network of factors may play a role in regulating hematopoietic cell survival. Surprisingly,

overexpression of murine Eya2 or human EYA2 induced rapid apoptosis that overrides the survival

functions provided by serum and IL-3. Similarly, inducible expression of the fly eya gene also

triggered rapid cell death, demonstrating that this apoptotic pathway is conserved from arthropods

to vertebrates. Moreover, biochemical and genetic data indicate that the major target of the Eya-

induced cell death pathway is mitochondria. A model for how this apoptotic pathway may function

during normal development is proposed.


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EXPERIMENTAL PROCEDURES

RT-PCR - mRNA was extracted from exponentially growing cultures of 32D.3 cells or the

developing mouse eye (E10.5) using Qiagen’s Direct mRNA Mini kit according to the manufacturer’s

protocol. Reverse transcription was performed with Qiagen’s Sensiscript RT kit per the manufacturer’s

directions. PCR was performed using a DNA thermal cycler (MJR Research, Waltham, MA). PCR

reactions contained 2µl of the cDNA product, 1x reaction buffer, 1.5 mM MgCl2, 2 mM dNTP, 20 pmol of

each primer, and 2.5 units of Taq polymerase (Perkin Elmer, Oakridge, TN) in a total volume of 25 µl. The

GAPDH primers were provided courtesy of Dr. Rachid Drissi (St. Jude Children's Research Hospital). The

primers used for RT-PCR were as follows: Eya1, sense primer 5’-CTAACCAGCCCGCATAGCCG-3’,

anti-sense primer 5’-TAGTTTGTGAGGAAGGGGTAGG-3’; Eya2, sense primer 5’-

CTCGCAACAAGCAGTGACTGG-3’, anti-sense primer 5’-GGACGGATAATCCTGGTGCAC-3’; Eya3,

sense primer 5’-AGCACAAATGCCAGCCTGATAC-3’, anti-sense primer 5’-

CTTCCACATGCACCTGGTCAC-3’; Pax6, sense primer 5’-GGGCAGGTATTACGAGACTGG-3’, anti-

sense primer 5’-GAGACAGGTGTGGTGGGCTG-3’; and GAPDH, sense primer 5-

GGTGAAGGTCGGTGTGAACGG-3’, anti-sense primer 5’-GTGGTGCAGGATGCATTGCTG-3’.

Cloning of human EYA2 - To isolate a human homolog of Drosophila eya, a partial human clone

(HPBEJ79) from an EST hemangioma cDNA library was obtained from Human Genome Sciences. This

EST contained an insert of 1445 nucleotides with the 5'-most 718 bases being 65% identical to Drosophila

eya. To acquire a complete cDNA, 5'-RACE (Rapid Amplification of cDNA ends, Life Technologies, Inc.)

was employed using mRNA from the human neuroblastoma cell lines LA-N-5 and CHP-134 (both obtained

from ATCC) which had abundant levels of EYA2 transcripts as determined by Northern blotting (Fee et al.,

in preparation). Three sequential 5'-RACE reactions were required to retrieve the entire EYA2 cDNA. Three

partial cDNA clones, one from each set of 5'-RACE extensions, were ligated by standard cloning procedures

to generate a complete EYA2 cDNA, which is predicted to encode a protein of 538 amino acid residues.


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Cloning of murine Eya2 - Based on areas of high homology between human EYA2 and eya, EYA2

primers were used for isolation of murine Eya2; sense primer, 5’-GGGACATTTGCATCCAGATAC-3’ and

anti-sense primer, 5’-AGCTTCCTCATCCAGTCCAC-3’. RT-PCR was performed as described above and a

300 base pair fragment was isolated from a murine T-cell leukemia cell line (RL-12). To extend the 3’-end

of the murine cDNA, 3’-RACE was performed as described by the manufactures protocol (Life

Technologies, Inc. Rockville, MD). To extend the 5’-end of the murine cDNA, 5’-prime RACE was

performed (Life Technologies, Inc.). Briefly, first strand cDNA synthesis was performed using a gene-

specific antisense oligonucleotide (2.5 pmoles), sample mRNA (500 ng) and SuperScript II reverse

transcriptase. The mRNA template was then degraded with RNase H and the purified cDNA was tailed with

dCTP and TdT (producing a 3’-(dC)n tail). The dC-tailed cDNA was then amplified by PCR using an anchor

primer (to dC tail) and a gene-specific nested primer. In addition to 5’-RACE, we searched the EST database

at NCBI and identified an EST which was identical to the 5’-end of our cDNA. Based on the sequence of

this EST and PCR we isolated this EST and with 5’-RACE we extended it until the Eya2 cDNA contained

the 5’-most ATG and several upstream in-frame stop codons.

Establishment and maintenance of inducibly-expressed Eya 32D.3 cells - A sequence encoding the

flag epitope (MDYKDDDDK) (Sigma, St. Louis, MO) was attached to the 5’-end of fly eya, murine Eya2

and human EYA2 cDNAs (the eya cDNA was kindly provided by Dr. Seymour Benzer, Cal Tech, CA). This

was accomplished with primers designed to include 7 base pairs (CAAGGCA) 5’ of the Eya2 ATG and the

flag sequence in frame with Eya2. High fidelity PCR was then performed using Expand Taq Polymerase

(Boehringer Mannheim, Indianapolis, IN). DNA sequencing was performed to ensure no mistakes were

generated during PCR. The tagged Eya2s and fly eya were then cloned into the XhoI (blunted) site of the

dexamethasone-inducible vector pMAM-Neo (Clontech, Palo Alto, CA).

32D.3 cells were maintained in RPMI 1640-10% fetal calf serum-1% L-glutamine medium

supplemented with 20U of IL-3 as described previously (24). Parental 32D.3 cells were electroporated by

washing the cells (90% viable) with RPMI (without additives), adding 20 µg of the linearized flag-tagged

pMAM-Neo-Eya expression plasmids into the electroporation chamber and mixing with 1 ml of cells (at 5 X


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106 cells/ml, refs. 23, 24). The chamber was then placed into a cell electroporator and electroporated with

the following conditions: 1180 µF, 375 volts and low resistance. Transfected cells were selected in medium

containing G418 and individual clones were isolated as described previously (23, 24). Control clones

containing the vector pMAM-Neo were isolated in parallel.

Prior to the start of all experiments, cells were set twice at 0.5 X 106 cells per ml on consecutive days

to ensure that they were in equivalent exponential phase of their growth. To induce expression of the Eya

proteins, clones were treated with 1.5 µM dexamethasone (dex) (Sigma).

Eya localization by confocal imaging - Cytospins were made of Eya2/eya clones with or without dex

(100µl of cells at 0.5 X 106 cells/ml). The slides were then fixed in 10% buffered formalin (Fisher) for 10-20

minutes at room temperature and the cells permeabilized with cold acetone for 2 minutes. The slides were

then washed with 1X PBS for 5 minutes and blocked in 10% BSA/1X PBS for 30 minutes at room

temperature. Primary flag antibody (M2, Sigma) was diluted 1:1000 in 10%BSA/1X PBS and placed on the

slides for 2 hours at room temperature. The slides were then washed three times with 1X PBS (5 minutes

each). The slides were incubated for 2 hours with fluoroscein-conjugated secondary antibody diluted 25-50

fold in 10% BSA/1X PBS. Following three washes with 1X PBS the slides were mounted with 0.1% p-

phenylenediamine in 1:9 PBS: glycerol before microscopic examination.

Confocal imaging was performed using a Leica DM-IRBE microscope together with Leica TCS-NT

software and equipped with a Nikon 100X, 1.4 N.A. objective and an argon/krypton laser (λ= 488/565), to

excite fluoroscein isothiocyanate (FITC) and Texas Red fluorescence.

Apoptosis assays - The viability of vector-only and Eya2/eya cells cultured in IL-3 growth medium

was assessed following treatment with or without dex, using a hemocytometer and trypan blue dye exclusion

as an indicator of cell viability. Cell morphology was assessed following cytospins and staining with

Wright-Geimsa.

Mitochondrial membrane potential was assessed by loading dex treated and untreated cells with

either 3,3-dihexyloxacarbocyanine iodide [DiOC6 (250 nM)] or tetramethylrhodamine ethyl ester [TMRM


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(500 nM)] (Molecular Probes, Eugene, OR) for 30 minutes. The cells were then washed twice in growth

media and then resuspended in growth media containing 100 nM of the respective dye. The cells were

incubated for 2 hours, washed in PBS and then analyzed for fluorescence changes by flow cytometry

(DiOC6) or confocal microscopy (TMRM) (25, 26).

Early plasma membrane changes were studied by using an Annexin V-FITC kit (Boehringer

Ingelheim Bioproducts). Cells were washed in PBS, resuspended in binding buffer (10 mM HEPES- NaOH,

pH. 7.4; 140 mM NaCl; 2.5 mM CaCl2) and cell density was adjusted to 5.0 x 105/ml. To 190 µl of cells, 10

µl of Annexin V-FITC was added and incubated for 10 minutes in the dark. The cells were then washed in

binding buffer and 10 µl of 20 ug/ml propidium iodide (PI) was added and the sample was analyzed by

fluorescence activated cell sorter (FACS) analysis.

The analysis of genomic DNA fragmentation was carried out as previously described (23). Briefly,

1.0 x 106 cells were washed with PBS, pelleted, resuspended in 20 µl of buffer (10 mM EDTA, pH 8.0; 50

mM Tris-HCl, pH 8.0; 0.5% sodium laurylsarcosine; 0.5 mg/ml proteinase K) and incubated for 1 hour at

50°C. DNA loading buffer was added, the samples incubated for 2 minutes at 70°C, loaded into the dry

wells of a 2% agarose gel (0.1 µg/ml ethidium bromide), run at 20 volts overnight (in TBE) and the gel was

photographed.

Electron micrographs were performed by the St. Jude Children’s Research Hospital EM facility

using standard protocols.

z-VAD.fmk (Sigma, St. Louis, MO) was dissolved in DMSO and used at a final concentration of

400µM, which effectively delays death of 32D.3 cells deprived of IL-3 (25). As a control, equal volume of

the vehicle DMSO was added to cells.

Cell cycle analyses - Cells were collected by centrifugation and resuspended in 0.1% sodium citrate

with 50 µg per ml of propidium iodide and analyzed as previously described (23, 24).

Western blot analysis - Cells were harvested and 75 to 100 µg of proteins were loaded per lane and

resolved by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were


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transferred to nitrocellulose using a semi-dry transfer cell (20 volts for one hour, Trans-Blot SD, Bio-Rad).

Following transfer, blots were incubated in a Fast Green staining solution (0.1% Fast Green [Sigma F7258],

20% methanol, 5% acetic acid) for 2 minutes and destained for 5 minutes (20% methanol, 5% acetic acid).

The blots were then washed in TBS-Tween three times for 10 minutes and blocked for one hour (5% non-fat

dry milk, TBS-Tween). The primary antibody was then diluted in blocking solution and placed on the blots

for one hour. Three washes followed in TBS-Tween for 10 minutes each. Horseradish peroxidase

conjugated secondary antibody was then added in blocking buffer for 30 minutes to each blot and then

washed in TBS-Tween as stated above before luminescence detection (SuperSignal, PIERCE, Rockford, IL).

The primary antibodies used were the following: Caspase-9 (Calbiochem, San Diego, CA), Caspase-3

(Pharmingen), PARP (Oncogene Research, #C-2-10, Boston, MA), anti-β tubulin (Oncogene Research), Flag

M2 (Sigma), Bad (Transduction Laboratories, #B36420), Bak (Santa Cruz, #G-23, Santa Cruz, CA), Bax

(Pharmingen, #13686E), Bcl-2 (Pharmingen, #15021A), Bcl-XL (Transduction Laboratories, #B22620) and

Mcl-1 (Transduction Laboratories, #M54020).

RESULTS

The Eya family is expressed in hematopoietic cells By screening cDNA libraries from

Human Genome Sciences, we identified a human homolog of Drosophila eya, EYA2, from a

hemangioma library (clone HPBEJ79). Using this EYA2 cDNA, we probed multiple tissue blots of

fetal murine tissue. These results revealed that in addition to its expression in the eye, Eya2 is also

expressed in E15.5 fetal brain, lung and liver (data not shown). The fetal liver is the principal site

of definitive hematopoiesis at this stage of murine development (26). These results suggested that

the collection of eye developmental regulators may also play a role in the development and/or


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survival of hematopoietic progenitors. We therefore assessed expression of the Eya family and

Pax6 in IL-3-dependent 32D.3 myeloid cells, which share many properties of primary

hematopoietic progenitors. In particular, 32D.3 cells continuously require IL-3 for their survival

(23). Surprisingly three of the four murine Eya members (Eya1, Eya2 and Eya3) were expressed in

exponentially growing 32D.3 cells and their expression was not dependent upon IL-3 (Fig. 1). By

contrast, Pax6 expression was not detected in 32D.3 cells, although it was detected in RNA isolated

from the developing murine eye (Fig. 1).

Eya2 and eya trigger apoptosis of murine 32D.3 myeloid cells To determine if Eya

proteins could influence the survival of 32D.3 cells, we performed 5'-RACE of the partial human

EYA2 cDNA from a Human Genome Science clone (HPBEJ79) to obtain a full-length cDNA

(GenBank Sequence Y10261) and then cloned the full length murine Eya2 from RNA prepared

from an Eya2-expressing murine T-cell leukemia cell line (RL-12) by RT-PCR and 5'- and 3'-

RACE (GenBank sequence BC003755). We then flagged-tagged fly eya, murine Eya2 and human

EYA2. The flag-tagged Eyas were then cloned into a pMAM-Neo vector and stable clones were

generated in 32D.3 cells following electroporation and growth in G418 and cloning by limiting

dilution. The pMAM-Neo vector is dexamethasone (dex) inducible, and after induction with dex

we detected proteins for eya, murine and human Eya2 at 95, 64 and 70 kilodaltons (kDa),

respectively (Fig. 2A). The predicted molecular weights for these proteins are 80 kDa (eya), 58

(murine Eya2) and 59 kDa (human EYA2) (15, 27). This suggests that all Eya proteins may be

post-translationally modified.

Eya proteins have been variably reported to be nuclear or cytosolic (1, 27, 28) and Eya

localization has been suggested to be regulated by interactions with the sine oculis/Six homeobox

family of proteins (15). We therefore assessed murine Eya2 localization by confocal microscopy


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following induction with dex. Eya2 was localized to both the cytoplasm and nucleus, yet was

excluded from the nucleolus (Fig. 2B). Similar results were also evident in clones that inducibly

express fly eya and in cells that overexpress human EYA2 (data not shown). The nuclear

localization of a significant fraction of Eya2 is consistent with its generally accepted function as a

regulator of transcription (11, 29).

To assess the role of fly eya and murine and human Eya2 in regulating 32D.3 cell survival,

vector only and Eya-expressing clones were treated with dex and then deprived of IL-3. As a

control, cells were also cultured in medium containing both IL-3 and serum and treated with dex.

Surprisingly, human and murine Eya2-expressing clones rapidly died following the addition of dex

even when these cells were cultured in replete IL-3 growth medium containing the full complement

of survival factors (Fig. 3). Similarly 32D.3 cells engineered to express fly eya also rapidly died

following the addition of dex, whereas (as expected) dex had no effect on the survival of parental

32D.3 cells or vector-only expressing cells (Fig. 3). Thus, eya and Eya2 overexpression induces cell

death and this pro-apoptotic function is conserved between fly, mouse and human proteins.

Eya loss-of-function mutants in the fly exhibit excessive proliferation, suggesting that eya

may regulate the cell cycle (11). Thus, we also measured whether the induction of Eya expression

led to changes in the cell cycle distribution in exponentially growing cultures or in cells deprived of

IL-3, a condition under which 32D.3 cells arrest in G1 (23). No obvious changes were evident in

Eya-overexpressing clones (data not shown), suggesting that Eya-induced cell death was not a

consequence of overt influence on cell cycle traverse.

Eya-induced cell death is apoptotic To assess whether eya-induced death was apoptotic

in nature, we analyzed both the DNA integrity and the ultrastructure of eya and Eya2 expressing

cells before and after treatment with dex. A hallmark of apoptosis is the condensation of chromatin


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into “micronuclei” and the cleavage of genomic DNA into oligonucleosomal units, resulting in a

“DNA ladder” (30). Following dex treatment, there was obvious internucleosomal DNA cleavage

occurring in Eya2 clones (Fig. 4A) and Wright-Geimsa staining of cytospins of dex-treated Eya-

expressing clones revealed typical condensation of chromatin (Fig. 4B). DNA condensation, and

condensation of the cytosol, were also apparent by electron microscopy (Fig. 4C).

Another hallmark of apoptosis is the flipping of phosphatidylserine residues from the inner

to the outer leaflet of the plasma membrane (31). Annexin-V binds to exposed phosphatidylserine

residues and FACS analyses can identify early apoptotic cells as those that stain with Annexin-V

but exclude propidium iodide (PI) as the cell membrane is intact. FACS analysis demonstrated that

dex treatment of Eya2 expressing clones, but not vector-only clones, led to a substantial increase in

Annexin-V-positive/PI-negative early apoptotic cells (Fig. 4D). Thus, we conclude that eya-

induced death of 32D.3 cells displays many of the hallmarks of apoptosis.

Caspase-3 is activated during, but is not essential for, Eya2- and eya-induced death.

Oligonucleosomal DNA fragmentation is mediated by DFF40, which is activated following

caspase-3-mediated cleavage of its inhibitor DFF45 (32-34). Activation of caspases involves a

cascade that first cleaves and activates initiator caspases, such as caspase-9, which then cleave and

activate downstream effector caspases, including caspases-3, -6, and –7 (35). Once activated

caspase-3 cleaves key targets required for cell integrity, including DFF45 and poly-ADP ribose

polymerase (PARP). We examined eya clones for caspase activation following the addition of dex

using antibodies specific for caspase-9, caspase-3, and PARP. Immunoblot analyses demonstrated

cleavage of caspase-9, caspase-3, and PARP following either eya or Eya2 induction (Fig. 5A).

Therefore eya-induced apoptosis involves caspase activation.


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To confirm that caspase activation was required for eya-induced apoptosis, we assessed

whether the broad-spectrum caspase inhibitor, z-VAD.fmk (36), could block the death of Eya

clones treated with dex. In parallel we evaluated effects of z-VAD.fmk on the death of parental

32D.3 cells deprived of IL-3. As expected (25), z-VAD.fmk effectively delayed 32D.3 cell death

associated with IL-3 withdrawal (Fig. 5B). However z-VAD.fmk failed to protect 32D.3 cells from

Eya2-induced death (and actually slightly accelerated their death, Fig. 5B), despite the fact that it

abolished DNA cleavage in these cells (Fig. 5C). Thus, Eya-induced apoptosis involves both

caspase-dependent and caspase-independent pathways.

Eya-induced apoptosis targets mitochondria Mitochondrial changes are associated with

many forms of apoptosis, including caspase-independent pathways (37). A common mitochondrial

alteration are reductions in membrane potential (∆ψ) (38, 39) and we therefore assessed whether

Eya2 expression influenced mitochondrial function by altering its ∆ψ. Initially we utilized the

fluorophore DiOC6, which is taken up by mitochondria and other organelles of viable cells, but

following changes in ∆ψ diffuses out of the mitochondria and cell (40). Eya2 clones cultured in

medium with and without dex were loaded with DiOC6 and analyzed 14 hours later by flow

cytometry (Fig. 6A). 32D.3 cells grown in medium with and without IL-3 were used as a negative

and positive control, respectively, for alterations in ∆ψ (Fig. 6A). After treatment with dex, 40% of

Eya2 clones underwent rapid decreases in membrane potential, whereas only 5% of untreated cells

exhibited overt changes in their membrane transition state. To address if this reduction in

membrane potential was specific to mitochondria, we used the fluorophore TMRM, a

potentiometric fluorescent dye that incorporates into mitochondria of viable cells in a ∆ψ dependent

manner (41). Eya2 clones and control cells in growth medium were cultured with and without dex,

loaded with TMRM and then observed by confocal microscopy (Fig. 6B). Again, dex-treated Eya2


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clones underwent a marked decrease in their ∆ψ, visualized by a decrease in the fluorescence of

their mitochondria, whereas these changes were not evident in control cells or Eya2 clones not

treated with dex (Fig. 6B). Therefore, Eya-induced apoptosis is associated with marked alterations

in ∆ψ.

Bcl-2 family members are known to regulate apoptosis in part through their effects on

mitochondrial function (42). Cytokines suppress apoptosis of myeloid progenitors at least in part

through their ability to selectively regulate the expression of the anti-apoptotic protein Bcl-XL (43)

and c-Myc-induced apoptosis in 32D.3 cells is associated with the selective repression of Bcl-2

expression (44). We therefore assessed whether Eya-induced apoptosis was associated with

alterations in the levels of anti- or pro-apoptotic Bcl-2 family members by immunoblot analyses.

However, the induction of Eya by dex did not result in the reduction of any of the anti-apoptotic

proteins studied and also failed to induce the pro-apoptotic members Bax, Bak or Bad (Fig. 7).

Caspase-dependent and -independent cell death is blocked by Bcl-2 overexpression (45) and

we therefore tested whether Bcl-2 overexpression could antagonize eya-induced death. Clones of

32D.3 cells engineered to overexpress human Bcl-2, which have a marked survival advantage when

deprived of IL-3 (44), were electroporated with the murine Eya2 expression construct and pools

overexpressing both Bcl-2 and Eya2 were identified by immunoblot analyses. These cells were then

analyzed for their viability following the induction of Eya2 with dex. Bcl-2 overexpression had no

effect on the induction of Eya2 protein by dex (data not shown) but effectively blocked Eya2-

induced apoptosis (Fig. 8). Overall these data suggest that Bcl-2 overexpression prevents Eya-

induced apoptosis by protecting mitochondrial functions.


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DISCUSSION

The role of the conserved class of transcription factors eyeless, eyes absent, sine oculis and

dachsund in development is well documented (2, 3, 45). Loss-of-function mutants and ectopic

overexpression studies in the fly have linked these proteins to the regulation of programmed cell

death, yet their direct role as activators of the apoptotic program have heretofore been lacking.

Here we have shown that, at least when overexpressed, eya can directly activate the apoptotic

program in 32D.3 myeloid cells and that this function is conserved between the fly, mouse and

human Eya proteins. 32D.3 cells express Eya1-3 and are a useful model to evaluate apoptotic

pathways as, like normal hematopoietic progenitors, they continuously require IL-3 to inhibit the

endogenous apoptotic program (23). The model originally proposed by Benzer and colleagues

suggested that eya may function as an anti-apoptotic protein (1), and therefore an expectation of our

studies was that Eya overexpression would inhibit the death of 32D.3 cells when they were deprived

of IL-3. Surprisingly, we found the opposite to be true and that eya acted as a pro-apoptotic signal

in this cell context. In part this is perhaps not entirely unexpected, as an imbalance of this

regulatory network in the fly does lead to inappropriate apoptosis (1, 4-7, 12, 13, 46). Overall our

data and those of others suggest that when one of the genes of this network is overexpressed that a

default apoptotic program is triggered. This death program could simply be the result of

inappropriate stoichiometry of these factors or could be due to a complete misregulation of eya

target genes.

To define how eya provoked the cell death program we examined biochemical changes

associated with its cell death program. Eya-induced apoptosis was associated with many of the


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hallmarks of typical apoptosis, including condensation of the cytosol and nucleus, plasma cell and

mitochondrial membrane changes, the activation of caspases and the degradation of genomic DNA

into a typical oligonucleosomal ladder. However, eya-induced death was not inhibited by the broad

caspase inhibitor z-VAD.fmk. It has been suggested that other proteases such as calpain can be

activated directly during apoptosis (45), or following the release of pro-apoptotic factors from

mitochondria such as apoptosis-inducing factor (38), and this may explain the failure of z-VAD.fmk

to rescue some forms of apoptosis (45). Consistent with what others have observed in caspase-

independent pathways, we did see extensive cell membrane blebbing in Eya-expressing cells in the

presence of z-VAD.fmk (data not shown) even though internucleosomal cleavage of genomic DNA

was blocked. Furthermore, eya-induced death was not associated with overt changes in the

expression of any Bcl-2 family members. However, Eya-induced apoptosis was blocked by the

overexpression of Bcl-2, which is consistent with Bcl-2’s ability to block both caspase-dependent

and independent death (42, 45). Thus, eya appears to induce apoptosis by triggering both caspase-

dependent and independent pathways. One unresolved issue is whether Eya somehow results in

changes in the localization of Bcl-2 family proteins. For example, Bax translocates from the

cytoplasm to the outer mitochondrial membrane following receipt of apoptotic signals (42) and this

could be occurring during eya-induced apoptosis. Furthermore, we suspect since most intrinsic cell

death signals appear to require the combined functions of Bax and Bak (47) that Eya-induced death

would have similar constraints.

It has been suggested that the cell deaths that occur in Drosophila eya mutants is due not to

their inability to control death but rather to their failure to properly control proliferation (11). This

concept is supported by excessive proliferation in the eye disc in these mutants, and the fact that cell

death is only observed following traverse of the morphogenetic furrow (11). By contrast, in 32D.3


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cells eya and Eya2 overexpression triggers apoptosis without overt effects on cell growth and cell

cycle traverse. Of course the simplest interpretation is that Eya functions may be cell context

specific, but another is that the loss-of-function fly eya mutants display excessive proliferation as a

consequence of inappropriate survival of progenitor cells, due to the lack of Eya-induced apoptosis.

The Drosophila eye regulator eyes absent and murine Eya2 are both capable of inducing

apoptosis when ectopically expressed in eyeless mutants in vivo (9). Thus eya and other eye

regulators may perform dual roles in development by both promoting eye development and

activating cell death. We propose that proper development versus cell death is dependent on the

appropriate levels of the eye regulators. Specifically when eya genes (or others such as sine oculis

or dachshund) are overexpressed, or when other members of the regulatory network are not present

in the proper stoichiometry, a default pathway is activated that triggers apoptosis (Fig. 9). Again

this could be due to inappropriate regulation of target genes of this network. Such a pathway would

ensure that misguided development rarely occurs. Several lines of evidence support this concept.

Firstly, when overexpressed in a wild-type background, eya, eyeless, sine oculis and dachshund all

result in a reduction of the eye (46). These deficits are phenotypically similar to loss-of-function

mutants in each of these genes, and are again associated with massive cell deaths in the eye field (1,

5, 7, 8). Secondly, when eyeless is ectopically expressed in the wing disc of either eyes absent or

sine oculis mutant flies, an eye is not formed but rather abnormal structures caused by massive cell

death (12). Similar observations have been reported when dachshund is overexpressed in either the

leg or wing of wild type flies (13). Finally, we have shown that overexpression of eya or Eya2 in

32D.3 cells, which do not express Pax6 (Fig. 1), induces apoptosis and we have also shown that sine

oculis overexpression also triggers death of these cells (W.C. and J.L.C., unpublished results).

Thus, overexpression of these eye regulators seems in most cases sufficient to trigger apoptosis.


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Recent studies have also implicated eyeless/Pax6 in triggering vertebrate cell deaths. For

example, during Xenopus development, Pax6 expression in the neural fold stage strikingly overlaps

with TUNEL-positive cells (48). Furthermore, high-copy-number Pax6 transgenic mice have a

severe microphthalmia phenotype (49). Finally, it has been shown that PARP, the target of caspase-

3, is itself a regulator of Pax6 gene expression in the neuroretina (50). Together with findings

demonstrating that the eya genes require Pax6 for expression (15), this suggests that a feedback

loop exists in which Pax6, Eya2, and PARP regulate cell death in the developing neuroretina.

There is also ample evidence that reduction in expression of the eye master regulatory genes

also triggers apoptosis. In addition to the wealth of data in the fly, where mutations in either eya,

eyeless, sine oculis or dachshund correlate with excessive apoptosis (1, 5, 7, 8), deletion of Eya1 in

mice leads to defective otic vesicle formation and this is associated with excessive apoptosis (51).

Further, the knockout of the ski gene (a proposed dachshund family member) results in embryos

with excessive apoptosis during neurulation (52, 53). Lastly, analysis of mouse mutants defective

in Pax6 expression indicates abnormal apoptosis (54). Therefore, overexpression or a failure to

express eye master regulatory genes appears sufficient to trigger the cell death program. This

suggests that the relative amounts of these proteins must be tightly controlled for proper

development. We propose that the apoptotic pathway is activated only when the stoichiometry of

these regulators is distorted due to changes in tissue-specific expression, or following mutations that

inactivate or silence the expression of these genes or alter their function or regulation of their target

genes.


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Acknowledgements - We thank Dr. Seymour Benzer (California Institute of Technology) for the

generous gift of the fly eya cDNA, Human Genome Sciences for the human EYA2 EST cDNA clone

HPBEJ79, Yanjun Ma (St. Jude) for help in preparing the manuscript, Elsie White and Chunying Yang for

outstanding technical support and members of the laboratory for their suggestions. This work was also

supported by Cancer Center Support Grant CA-21765 and by American Lebanese Syrian Associated

Charities (ALSAC) of St. Jude Children’s Research Hospital.

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FIGURE LEGENDS

FIG. 1. Eya1-3, but not Pax6, are expressed in IL-3-dependent 32D.3 myeloid cells. Semi-

quantitative RT-PCR was performed on RNA isolated from 32D.3 cells cultured in IL-3 growth

medium or from 32D.3 cells deprived of IL-3 for 14 hours. As a control, RNA was also isolated

from day E10.5 of the developing murine eye. Expression analyses included Eya1, Eya2, Eya3,

Pax6 and GAPDH.

FIG. 2. Eya2/eya expression and Eya2 localization within 32D.3 cells. A, Protein (100 µg/lane)

from the indicated 32.D.3 clones treated with or without dex was isolated and resolved on a 12%

polyacrylamide gels and Western blot analysis was used to determine Eya2, EYA2 and eya

expression. Since Eya2, EYA2 and eya are flag-tagged, the primary antibody is anti-flag. B, To

determine Eya2 localization within 32D.3 cells, Eya2-expressing cells treated with dex (to induce

Eya2 expression) were fixed for immunohistochemistry and analyzed by confocal microscopy using

the anti-flag antibody (green stain). Propidium iodide (PI, red stain) was used to stain DNA in the

nucleus.

FIG. 3. Overexpression of Eya2 and eya in 32D.3 cells overrides the survival factors IL-3 and

serum to trigger cell death. Cultures of 32D.3 clones growing in IL-3 and serum and engineered

to inducibly express Eya2, eya, or the vector alone (Neo, electroporated at the same time as Eya2,

EYA2 and eya) were treated with dex for the indicated intervals. Cell viability was determined at

the indicated intervals following the addition of dex by trypan blue dye exclusion (n=3). When

deprived of IL-3 and treated with dex Eya-expressing clones displayed a marked acceleration in

their rates of death (data not shown).


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FIG. 4. Eya2 and eya overexpression induces apoptosis in 32D.3 myeloid cells. A, 1 x 106 Eya2-

expressing or Neo vector cells growing in IL-3 and serum were treated with the vehicle alone (-) or

were treated with dex for 24 hours (+). After 24 hours genomic DNA was prepared and loaded on a

2% agarose gel. As a positive control for internucleosomal DNA cleavage, genomic DNA was

isolated from 32D.3 cells overexpressing c-Myc and deprived of IL-3 (Myc.2-IL-3), which display

an accelerated course of apoptosis (23). B, Chromatin condensation was examined by making

cytospins of Eya2 and Neo clones stained with Wright-Geimsa 24 hours after dex treatment. C,

32D.3 clones expressing either fly eya or the Neo vector were treated with dex for 24 hours,

isolated and fixed for electron microscopy. The bar at the bottom of each picture denotes a size of

two microns. Note the condensed micronuclei in cells expressing fly eya. Similar results were

obtained in cells expressing Eya2 (data not shown). D, To examine phosphatidylserine flipping, a

clone that inducibly overexpressed Eya2, 32D.3/Eya2.5, was treated with or without dex for 10

hours, the cells washed with PBS and Annexin V was added. FITC-conjugated anti-Annexin V was

then added to the cells along with PI and the cells were analyzed by flow cytometry. Annexin V+,

PI– are early apoptotic cells (bottom right rectangle) that were 8-fold higher in Eya2 cells treated

with dex. Similar results were obtained in clones engineered to express fly eya and dex treatment

did not induce Annexin-V positive cells in vector-only control cells (data not shown).

FIG. 5. Caspase-3 is activated by, but is not essential for, Eya2- and eya-induced death. A,

Protein (100 µg) was isolated from the indicated cells treated with or without dex (14hrs) and run

on a 12% polyacrylamide gel. Western blot analysis was performed with flag (for Eya2 and eya

expression), caspase-9, caspase-3, PARP and tubulin antibodies. Both the proenzyme form

(Proform) and the cleaved product (Cleaved) are shown for caspase-9 and PARP, but only the


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cleaved form of caspase-3 is shown. Tubulin served as a loading control. B, The broad caspase

inhibitor z-VAD.fmk fails to protect 32D.3 cells from Eya-induced apoptosis. An Eya2-expressing

clone (Eya2.5) was treated with dex and the caspase inhibitor z-VAD.fmk (400 µM) for up to 24

hours. As a control, parental 32D.3 cells cultured in the presence or absence of IL-3 were incubated

with z-VAD.fmk. As shown, z-VAD.fmk protected 32D.3 cells from growth factor-withdrawal-

induced apoptosis, but was unable to inhibit Eya-induced death. Viability was determined as

indicated in Figure 3 (n=3). C, To confirm that the caspase inhibitor was still effective in Eya2-

expressing cells we assessed its ability to inhibit DNA cleavage. Cells were treated with dex for (20

hrs) and with or without z-VAD.fmk. DNA was isolated and run on a 2% agarose gel. L, DNA

size markers. As shown, z-VAD.fmk was able to inhibit DNA cleavage in Eya2-expressing cells.

Nonetheless, these cells still underwent cell death (B).

FIG. 6. Eya-induced apoptosis targets the mitochondria. A, To examine overall cell membrane

integrity, DiOC6 was loaded into 32D/Eya2.5 cells treated with or without dex (for 14 hours) and

washed to determine membrane leakage. Cells undergoing membrane changes will leak the dye and

their fluorescence intensity will decrease. The numbers indicate the number of cells that have a

decrease (a shift to the left) in their fluorescence intensity and thus decreases in their ∆ψ. As a

control, 32D.3 cells were grown in media containing IL-3 or were deprived IL-3, which induces

their apoptosis (23). B, To examine if the changes in ∆ψ were specifically associated with

mitochondrial changes, we examined mitochondrial ∆ψ changes with the dye TMRM (which is

mitochondria specific) in Eya2.5 cells treated (14 hrs) with or without dex. Cells were loaded with

TMRM (500 nM), washed in growth media, placed in growth media with 100 nM TMRM,

incubated for 2 hours, washed in PBS and prepared for confocal microscopy. Again, if


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mitochondrial ∆ψ decreased, the dye would not be retained by the mitochondria and a decrease in

fluorescence would occur. The hatched circle indicates the outline of the Eya2-expressing cells.

FIG. 7. Eya2- and eya-induced apoptosis is not associated with significant changes in the levels

of Bcl-2 family proteins. Protein was isolated from exponentially growing cultures of clones

expressing Eya2 or eya treated with or without dex for 14 hours. Western blot analysis was

performed with antibodies to the following Bcl-2 family members: Bad, Bak, Bax, Bcl-2, Bcl-XL,

and Mcl-1. Tubulin was used as a loading control and 100 µg of protein was loaded per lane. A

slight decrease in protein specific signal that was seen in most lanes treated with dex was found to

be due to the release of histones from DNA following cleavage (data not shown).

FIG. 8. Bcl-2 overexpression impairs Eya-induced apoptosis. To examine if overexpression of

Bcl-2 could inhibit Eya2-induced apoptosis, we electroporated 32D.3 cells overexpressing human

Bcl-2 (hBcl-2) with the inducible Eya2 expression construct. These parental 32D/hBcl-2 cells have

been previously shown to protect cells from IL-3 withdrawal-induced apoptosis (44). The viability

of a clone overexpressing both Bcl-2 and Eya2 (inset) was determined by trypan blue dye exclusion

in cultures treated with dex (n=3). 32D/Eya2 cells treated with dex are shown as a control.

FIG. 9. A proposed model for how overexpression of Eya family members leads to apoptosis

rather than eye development. A, When the eye regulators are expressed at their proper levels

normal development occurs. In this scenario target genes are correctly regulated. B, If the levels of

one of the eye regulators is either deficient or in excess a default apoptotic pathway is triggered,

which may be due to simple changes in stoichiometry of these factors and/or to alterations in the


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regulation of critical target genes. Arrows indicate feedback loops that exist within the network.

eyeless (ey) is required for the expression of eyes absent (eya), dachshund (dac) and sine oculis (so)

yet these factors can also regulate the expression of eyeless (1 and 2 in figure, refs 9, 13) and each

other.


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L + -

32D.3IL-3

E10

.5 E

ye R

NA

(-)

RN

A

Eya1

Eya2

Eya3

Pax6

GAPDH


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B.ααFlag PI Overlay

A. - + - + - + - +

Eya2.8Eya2.5Eya2.1 eya1.10 Dex

66 kDA

97 kDA

Eya2 eya

Tubulin

EYA2 pool - + - + Dex

EYA2

1 2


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eya1.10

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+ - + - + -

eya1.1 Neo.1

Neo.1 Eya2.5

+Dex- Dex

Neo.1 Eya2.1 Eya2.5

Myc

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Dex

Eya2.5


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C. L (+) (-) z-VAD.fmk

+Dex

B.

A. - + - + - + - +

Caspase-9

Caspase-3

PARP

Tubulin

Eya2/eya

Eya2.1 Eya2.5 Eya2.8 eya1.10

ProformCleaved

Cleaved

CleavedProform

Dex

10

20

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50

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0 14 24

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Eya2.5 (Dex + DMSO)Eya2.5 (Dex + z-VAD)32D-IL3 (Dex + DMSO)32D-IL3 (Dex + z-VAD)


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2% 39%-IL3+IL3

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32D.3


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Eya2.1 Eya2.5 Eya2.8

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Bad

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ey eya

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S. Wesley Clark, Brian E. Fee and John L. ClevelandMisexpression of the eyes absent family triggers the apoptotic program

published online November 7, 2001J. Biol. Chem.

10.1074/jbc.M108410200Access the most updated version of this article at doi:

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http://www.jbc.org/lookup/doi/10.1074/jbc.M108410200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M108410200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2001/11/07/jbc.M108410200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2001/11/07/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2001/11/07/jbc.M108410200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

Misexpression of the Eyes Absent Family Triggers the Apoptotic ...

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