rapid apoptosis induction by igfbp-3 involves an igf ... · rapid apoptosis induction by igfbp-3...

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Rapid apoptosis induction by IGFBP-3 involves an IGF -independent nucleo-mitochondrial translocation of RXRα/Nur77

Kuk-Wha Lee1, Liqun Ma1, Xinmin Yan1, Bingrong Liu1, Xiao-kun Zhang2 &Pinchas Cohen1*

1Division of Pediatric Endocrinology, Mattel Children's Hospital at UCLA, DavidGeffen School of Medicine, Los Angeles, CA, USA2Cancer Center, The Burnham Institute, La Jolla, CA, USA

Running title: IGFBP-3 translocates RXRα/Nur77

*To whom correspondence may be addressed. Tel: (310) 206-5844. Fax: (310)206-5843. E-mail: [email protected]

SUMMARY

Insulin-like growth factor binding protein-3 (IGFBP-3) induces apoptosis by its

ability to bind IGFs as well as its IGF-independent effects involving binding to

other molecules including the retinoid X receptor-α (RXRα). Here we describe

that in response to IGFBP-3, the RXRα binding partner nuclear receptor Nur77,

rapidly undergoes translocation from the nucleus to the mitochondria, initiating

an apoptotic cascade resulting in caspase activation within 6 hours. This

translocation is a type 1 IGF receptor signalling-independent event as IGFBP-3

induces Nur77 translocation in R- cells. IGFBP-3 and Nur77 are additive in

inducing apoptosis. GFP-Nur77 transfection into RXRα WT and KO MEFs and

subsequent treatment with IGFBP-3 shows that RXRα is required for IGFBP-3

induced Nur77 translocation and apoptosis. Addition of IGFBP-3 to 22RV1 cell

lysates enhanced the ability of GST- RXRα to “pull-down” Nur77, and

overexpression of IGFBP-3 enhanced the accumulation of mitochondrial RXRα.

This unique non-genotropic nuclear pathway supports an emerging role for

IGFBP-3 as a novel, multi-compartmental signaling molecule involved in

induction of apoptosis in malignant cells.

JBC Papers in Press. Published on February 24, 2005 as Manuscript M412757200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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INTRODUCTION

Over the past decade, multiple lines of investigation have validated Insulin-

like growth factor binding protein-3 (IGFBP-3) as an inducer of cellular apoptosis,

effects that can be unrelated to its IGF-binding (1). Importantly, several groups

have now reported successful in vivo treatment of cancer models with IGFBP-3,

either as a single-agent or in combination with chemotherapeutic agents (2-4).

However, the molecular mechanisms by which IGFBP-3 induces apoptosis

remain largely unknown at present.

Several novel IGFBP-3 binding partners have been recently identified that

may participate in its IGF-independent pro-apoptotic effects (1). We and others

demonstrated that retinoid X receptor-α (RXRα) is a binding partner for IGFBP-3

(5,6), and that RXRα is required for IGFBP-3 apoptotic effects (5). Indeed, IGFBP-

3 potentiates RXRE-mediated signalling while inhibiting signalling via other

RXRα heterodimeric partners (5-7). Our discovery of IGFBP-3 binding to RXRα

suggested that its apoptotic effects might involve a RXRα-dependent

transcriptional mechanism. Most published reports have evaluated IGFBP-3

induced apoptosis at 24 to 72 hours (8-11), consistent with a transcriptional

mechanism. However, we have recently described apoptosis activation by

IGFBP-3 (as evidenced by caspase activation and histone associated DNA

fragmentation ELISA) as early as 1-6 hours after IGFBP-3 exposure, suggesting

a mechanism that does not require de novo gene transcription (12,13).

The orphan nuclear receptor Nur77 (also known as NGFI-B (14) and TR3

(15)) is a nuclear receptor transcription factor and is an important regulator of


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apoptosis in different cells (16). It is a member of the orphan steroid receptor

family, which also includes Nor1 and Nurr1. This family is essential for

apoptosis of self-reactive immature thymocytes following stimulation of the T-cell

receptor (17,18). In response to synthetic apoptotic stimuli, Nur77 translocates

from the nucleus to the mitochondria to induce cytochrome c release and

apoptosis in leukemia (19), lung (20), ovary (21), stomach (22), colon (23), and

prostate cancer cells (24). Subcellular localization of Nur77 is important for its

biologic function. In the nucleus, it functions as a transcription factor to mediate

cell proliferation events. Targeted to the mitochondria, it takes on a novel role as

a mediator of apoptosis, not unlike the role played at the mitochondria by another

transcription factor, p53 (25). Importantly, Nur77 can also heterodimerize with

RXRα (26) and participate in its transcriptional activities (26-28). The mitogenic

effect of Nur77 requires its DNA binding and transactivation functions in the

nucleus whereas both are dispensable for the apoptotic effects of Nur77 at the

mitochondria (29).

Because the nuclear receptor RXRα is an intracellular binding partner for

IGFBP-3, we hypothesized that IGFBP-3 would modify RXRα/Nur77 heterodimeric

DNA binding, shifting this heterodimer from a DNA binding state to one that

targets mitochondria. Mitochondrial translocation of RXRα/Nur77 would then

result in the release of cytoplasmic cytochrome c, activation of intracellular

caspases, and induction of apoptosis.

Herein we report evidence that IGFBP-3 is a rapid biological signal

molecule for RXRα/Nur77 translocation. Our results reveal a new interaction


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between the nuclear receptor and IGFBP superfamilies and identify IGFBP-3 as a

unique signal modulator of both traditional and novel nuclear receptor roles at

the junction of cellular proliferation and apoptosis.


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EXPERIMENTAL PROCEDURES

Materials--Celtrix (Mountain View, California) provided recombinant human

IGFBP-3. IGF-1 was a generous gift from Pharmacia Corporation (Stockholm,

Sweden). Commercial antibodies included: anti-human IGFBP-3 from DSL

(Webster, Texas), anti-Nur77 from Geneka Biotechnology (Montreal, Canada),

anti-RXRα from Santa Cruz Biotechnologies (Santa Cruz, California), anti-

cytochrome c from Pharmingen (BD Biosciences, Palo Alto, California), and anti-

β-actin from Sigma-Aldrich (St. Louis, Missouri). PMP70 Antibody and cathepsin

S antibodies were from Zymed (South San Francisco, CA) and R & D systems

(Minneapolis, MN) respectively. For the western immunoblot utilizing the R-

MEFs, polyclonal rabbit anti-Nur77 antibody (Harlan Biosciences, Indianapolis,

Indiana) was generated against two specific N-terminal peptides (Genemed

Synthesis, San Francisco, California) derived from human Nur77 peptide

sequence. Sera was purified on protein A/G column (Amersham/Pharmacia,

Sunnyvale, California) and verified by Western blotting. Nur77 banding pattern

was confirmed using CCRF-CEM nuclear extract (Active Motif, Carlsbad,

California). SDS-polyacrylamide gel electrophoresis (PAGE) reagents, Tween,

and fat-free milk were purchased from Bio-Rad (Hercules, California). ECL

reagents were from Amersham (Sunnyvale, California). Full length IGFBP-3 and

Nur77 cDNAs was cloned into pLP-IRESneo mammalian expression vector via

pDNR-mediated CreatorTM technology (Clontech, Palo Alto, California). The

cloning of GFP-Nur77 has been described (24). LipofectAMINE and PLUS


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Reagent were from Invitrogen (Carlsbad, California). All other chemicals were

from Sigma-Aldrich.

Cell Culture--22RV1 cells, A172 cells, CCRF-CEM, and F9 embryonal carcinoma

cells from ATCC (Manassas, VA), and F9 RXRα -/- cells (kind gift of Dr. P.

Chambon) were maintained in Dulbecco’s modified Eagle’s medium containing

10% fetal calf serum (Life Technologies, Carlsbad, California), 100 units of

penicillin/ml, and 100 units of streptomycin/ml in a humidified environment with

5% CO2.

Mouse Embryonic Fibroblast (MEF) Generation--Fibroblasts from an IGF-I

receptor knockout and corresponding wild type mouse were generated from 18-

day embryos as described previously (30) and were designated R- and WT MEFs

respectively. The R- cells were maintained in Dulbecco's modified Eagle's

medium containing 10% fetal bovine serum and Geneticin (G418). All cells were

used before passage 6.

Apoptosis ELISA--Cells (2500 cells/cm2) were seeded on 96-well plates.

Following overnight attachment, cells were washed with PBS and serum starved

overnight, before incubation with the indicated conditions in a total of 100 µl

volume. Roche Photometric Cell Death ELISA (Indianapolis, IN) was performed

according to the manufacturer’s instructions to quantify histone-associated DNA

fragments (mono- and oligo-nucleosomes) generated by apoptotic cells. This


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immunoassay is based on the sandwich-enzyme principle and used separate

mouse monoclonal antibodies directed against DNA fragments and histones.

Briefly, cell lysates were placed into a 96-well plate coated with streptavidin-

linked, anti-histone antibody. Peroxidase-labeled mouse monoclonal DNA

antibodies were used to localize and detect the bound, fragmented DNA by

photometry of 2,2'-azino-bis-[3-ethylbenzathiazoline sulfonate] as the substrate.

Calcium ionophore treatment served as the positive control, and serum-free

medium (SFM) as the negative control. Each experimental condition was

performed in triplicate. Reaction products in each 96-well plate were read using

a Bio-Rad microplate reader. Mean absorbance data at 405 nm (±SD) were

plotted. Studies to provide evidence that the absorbance values generated by the

assay are linearly related to growth were performed and confirmed in a

published paper from our lab (31).

Caspase assays--The caspase assay was done using Apo-ONETM homogenous

caspase -3/-7 assay (Promega) and performed according to manufacturer’s

instructions. rhIGFBP-3 was used at a final concentration of 1 µg/ml.

Immunofluorescence Confocal Microscopy--Ten thousand cells were plated on

coverglasses in serum containing media for 2 days. Cells were then incubated

in serum-free media with or without IGFBP-3 before staining for

immunofluorescence. After three washes in phosphate-buffered saline (PBS),

fixation and permeabilization of the cells were performed with 1%


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paraformaldehyde in PBS for 15 min at room temperature and 0.2% Triton X-100

in PBS for 15 min on ice, and cells were washed twice with PBS. Nur77 or

IGFBP-3 protein localization was detected using hIGFBP-3/ Nur77 polyclonal

antibodies (diluted 1:1,000) followed by fluorescein/Texas Red antibody from

Vector Laboratories (Burlingame, California). Specimens were incubated with

primary antibodies in PBS for 1 h at room temperature, with secondary

antibodies in PBS for 40 min at room temperature, and then incubated with

Hoechst (Electron Microscopy Sciences, Ft. Washington, Pennsylvania) for 2

minutes. Samples were analyzed using inverted confocal microscopy (Leica,

Inc., Germany), equipped by digital camera Himamatsu (Japan), and operated by

QED-image software.

Subcellular fractionation Procedures--Nu-CLEAR Protein Extraction KitTM was

from Sigma-Aldrich. Subcellular fractions were isolated according to the

manufacturer’s protocol. The ApoalertTM Cell Fractionation Kit (BD Clontech) was

used to isolate a mitochondrial fraction from the cytoplasm of cells. Purity of the

fraction was assessed by immunoblot for PMP70 (peroxisomal) and cathepsin S

(lysosomal) contamination.

Transient Transfections--Cells (2 x 104) were seeded in 96-well culture plates.

Reagents were appropriately scaled up to 6-well plates for transfections that

were followed by mitochondrial isolation and subsequent western

immunoblotting. Transfections were done with LipofectAMINE:PLUS Reagent as


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directed by manufacturer (Invitrogen). Typically, 50 ng of β-galactosidase

expression vector (pSV-β-Gal, Promega, Madison, Wisconsin), and 50 ng

expression vector containing IGFBP-3 and/or Nur77 were mixed with carrier DNA

to give 0.2 mg total DNA per well. After 24-48 hours transfection, caspase activity

was quantitated and normalized for transfection efficiency to measurements of

aliquots of co-transfected β-galactosidase gene activity (β-galactosidase enzyme

assay system, Promega).

GST Pull-down--The GST-RXRα fusion vector encoding the full-length RXRα

molecule and was the generous gift of Dr. D. J. Mangelsdorf and has been

previously described (32). GST-RXRα fusion protein was produced in GST-

RXRα-transformed Escherichia coli DH5, which were lysed and loaded on

glutathione-sepharose 4B beads (Sigma). Ten µg of purified GST-RXRα bound

to beads was incubated with 500 µg of cell lysate, with or without 200 ng of

recombinant IGFBP-3 protein and then separated by centrifugation. The bound

proteins were analyzed by nonreducing SDS-PAGE followed by Western blotting

using anti- Nur77 antibody. Experiments were repeated three times.

Densitometric and statistical analysis--Densitometric measurement of

autoradiographs was performed using computer scanned densitometry. All

experiments were repeated at least three times. Means +/- SD are shown.

Statistical analyses were performed using ANOVA utilizing InStat (GraphPad, San


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Diego, California). Differences were considered statistically significant when p<

0.005, denoted by **.


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RESULTS

IGFBP-3 Induces Rapid Induction of Apoptosis—We have previously observed

that in A172 glioblastoma cells, IGFBP-3-induced caspase activation reaches its

maximum at one to six hours, after which it decreases (12). Likewise, in human

macrovascular umbilical vein endothelial cells, VEGF-induced survival of HUVEC

is inhibited by IGFBP-3, via the induction of apoptosis in a type 1 IGF receptor-

independent manner utilizing the neutralizing antibody αIR3 (13). We therefore

confirmed these rapid effects in a variety of cell lines. Mouse Embryonic

Fibroblasts (MEF) exhibited a 70% increase in apoptosis as evidenced by

fluorometric assessment of caspase 3/7 activation (Fig. 1A) as early as 2 hours

post treatment. This induction was maximal to nearly 2.5 fold over baseline at 6

hours. Similarly, in the human glioblastoma cell line A172, an almost 2-fold

increase in apoptosis was detected as early as one hour (Fig 1B). In the 22RV1

prostate cancer cell line (Fig. 1C), a significant 32% increase in caspase

activation was induced by the addition of IGFBP-3. This subsequently rose to a

40 and 51% increase over serum free levels at 6 and 24 hours respectively.

These results confirm that IGFBP-3 induction of apoptosis, assessed in multiple

cell lines, is a rapid event. Experiments were repeated three times.

IGFBP-3 Induces Rapid Nucleo-Mitochondrial Translocation of Nur77--To

investigate whether Nur77 translocation could mediate the pro-apoptotic effects

of IGFBP-3 in CaP cells, we first established that IGFBP-3 leads to nucleo-

mitochondrial translocation of Nur77. We performed a time course of IGFBP-3


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treatment and observed the subcellular localization of Nur77 by indirect

immunofluorescence confocal microscopy in 22RV1 CaP cells. Within 15

minutes there was strong cytoplasmic appearance of red-staining Nur77

compared to minimal cytoplasmic labeling at time 0 (Fig. 2A). The faint nuclear

staining seen at time 0 may reflect inaccessibility of anti-Nur77 antibody to the

protein secondary to RXR heterodimerization or Nur77 homodimerization. The

cytoplasmic Nur77 presence was maintained throughout this 2-hour time course

although some Nur77 reappeared in a nuclear location by the end of 2 hours.

To confirm that IGFBP-3 is a biologic Nur77 translocation signal, we also

assessed relative Nur77 concentrations in nuclear and cytoplasmic fractions of

IGFBP-3-treated 22RV1 CaP cells by Western immunoblot at 0, 0.5, 1, and 3

hours (Fig. 2B). Cells treated with IGFBP-3 showed the gradual disappearance

of nuclear Nur77 over the course of 3 hours associated with some increase in

the amount of cytoplasmic Nur77. Furthermore, a dramatic increase was shown

to be via mitochondrial targeting as subdivision of the cytoplasmic fraction into a

mitochondrial enriched fraction revealed the appearance of a prominent Nur77

band as detected by Western immunoblot as early as 1 hour (Fig. 2C).

Membranes were probed with PMP70 and cathepsin S to show that the isolated

mitochondrial fraction was free of peroxisomal / lysosomal contamination.

Similar results were obtained for the LAPC-4 prostate cancer cell line and A172

glioblastoma cell line (data not shown). A representative of three separate

experiments is shown.


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Rapid Mitochondrial Translocation of Nur77by IGFBP-3 occurs via a type 1

receptor independent mechanism--The possibility that IGFBP-3 acts to induce

apoptosis independently of IGFs and IGF receptors was investigated by testing

the ability of IGFBP-3 to induce apoptosis in the IGF receptor-negative (R-)

embryonic fibroblast cells derived from an IGF-1R knockout mouse (30). This

effect is mediated in part by a type 1 IGF receptor independent mechanism as

IGFBP-3 was still able to induce apoptosis, with a 32% increase over baseline at

2 hours, that was maximal at 6 hours in type 1 IGF receptor disrupted MEFs in a

fragmented DNA/histone ELISA (Fig. 3A). These cells have been shown

previously to neither bind nor respond to IGFs. IGFBP-3 induced mitochondrial

translocation of Nur77 in this unique system was further demonstrated by

immunoblotting analysis, which showed accumulation of Nur77 in the

mitochondrial fraction (Fig. 3B). To demonstrate the purity of the mitochondrial

fraction, expression of mitochondrial-specific protein Hsp60 and nuclear-specific

protein poly (ADP-ribosyl) polymerase (PARP) is shown as well as immunoblots

to PMP70 and cathepsin S (peroxisome/lysosome markers respectively). This

data suggests that Nur77 mitochondrial translocation by IGFBP-3 occurs

independent of signalling via the type 1 receptor. This experiment was repeated

three times.

Additive apoptotic effects of overexpression of IGFBP-3 and Nur77--To determine

whether mitochondrial targeting of Nur77 by IGFBP-3 plays a role in regulating

the release of cytochrome c from mitochondria into cytosol, the location of


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cytochrome c was examined during the course of IGFBP-3 treatment.

Immunoblotting of a cytoplasmic fraction that was depleted of mitochondria

showed that the addition of 1 µg/ml of IGFBP-3 caused a greater than 3-fold

increase in the appearance of cytoplasmic cytochrome c at 30 minutes, which

increased to almost 6-fold at 60 minutes that was sustained at 180 minutes (Fig.

4A).

The release of cytoplasmic cytochrome c is directly upstream of caspase

activation in the mitochondrial pathway of apoptosis (33). Overexpression of

Nur77 in thymocytes induces massive apoptosis (34). We co-expressed by

transient transfection both IGFBP-3 and Nur77 in mammalian expression vectors

to assess effects on caspase activation in 22RV1 cells. Expression of both

IGFBP-3 and Nurr7 in combination resulted in additive effects on caspase

activation (Fig. 4B), indicating additivity between IGFBP-3 and Nur77 in their

apoptotic effects. Protein expression was confirmed with immunoblots of whole

cell lysates from transfected cells (Fig. 4C).

IGFBP-3 induced Nur77 Translocation is RXRα-dependent, and involves co-

migration of RXRα/Nur77 heterodimers to mitochondria--We previously reported

that the nuclear receptor RXRα is a nuclear binding partner for IGFBP-3 and is

required for IGFBP-3 induced apoptosis (5). Also, in response to nerve growth

factor (NGF), nuclear RXRα/Nur77 heterodimeric complexes translocate to the

cytoplasm in PC12 pheochromocytoma cells (35). A recent paper also describes

the carrier role of RXRα to assist Nur77 translocation in the 9-cis retinoic acid-


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dependent apoptosis of gastric cancer cells (36). We hypothesized that pro-

apoptotic IGFBP-3 would translocate RXRα/Nur77 heterodimers to the

mitochondria. To investigate the role of RXRα in IGFBP-3-induced Nur77

translocation, F9 RXRα+/+ (wild-type, WT) embryonal carcinoma cells and F9

RXRα-/- cells were treated with 1 µg/ml IGFBP-3 overnight, nuclear fractions were

isolated, resolved via SDS-PAGE, and immunoblotted for the presence of Nur77.

Bands were quantitated by densitometric analysis and normalized to nuclear

PARP. As expected, IGFBP-3 induced a >50% reduction of nuclear Nur77 in the

wild-type cells, consistent with an RXRα/Nur77 translocation event (Fig. 5A). In

contrast, treatment of the sister RXRα-/- line with IGFBP-3 resulted in an increase,

rather than decrease, in nuclear Nur77. To confirm this observation, we

transfected green fluorescent protein (GFP)-Nur77 in the same cell lines, treated

with IGFBP-3, and visualized these cells utilizing confocal microscopy.

Expression of GFP-Nur77 in the RXRα WT line showed a predominantly nuclear

distribution consistent with its function as a transcription factor at basal

conditions (Fig. 5B). Treatment with 1 µg/ml of IGFBP-3 resulted in the rapid

appearance of extranuclear GFP-Nur77 observed within 15 minutes. This effect

was not seen in the sister RXRα-/- line, as IGFBP-3 treatment has no effect on the

translocation of nuclear GFP-Nur77. In addition, transfection of the Nur77

overexpression vector into F9 RXRα+/+ cells induced a marked increase in

caspase activation while transfection into the sister F9 RXRα-/- line failed to

induce any significant increase in caspase activity (Fig. 5C). Indeed, addition of

200 ng of IGFBP-3 to 500 µg of 22RV1 cell lysate demonstrated that IGFBP-3


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enhanced the ability of GST-RXRα to “pull-down” Nur77 (Fig. 5D), indicating that

IGFBP-3 augments the ability of RXRα/Nur77 to physically associate. In fact,

isolation of mitochondria from cells transfected with IGFBP-3 revealed a 3-fold

increase in mitochondrial RXRα (Fig. 5E) that was not seen in cells transfected

with control expression vector, demonstrating that IGFBP-3 leads to the co-export

of RXRα and Nur77. Together, our results demonstrate that the mechanism of

IGFBP-3 induced Nur77 translocation, like IGFBP-3-induced apoptosis, requires

RXRα, and involves co-migration of RXRα/Nur77 heterodimers to mitochondria.


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DISCUSSION

Multiple lines of in vitro, in vivo, and clinical evidence point to IGFBP-3 as

an anti-cancer molecule (37). In vitro, IGFBP-3 has been shown to induce

apoptosis in a variety of cancer models via both IGF-dependent and

–independent mechanisms (1). In vivo, recent publications report efficacy either

as single agent or in chemotherapy combinations in non-small cell-lung cancer

(2) and colon cancer (3). IGFBP-3 gene expression is commonly lost in human

cancer cell lines and xenografts as detected in DNA microarray analysis of

cancerous compared with non-cancerous cells (38). Decreased IGFBP-3

expression is associated with prostate cancer progression, demonstrating more

frequent loss of expression in advanced disease, in both human (39) and mouse

(40) models. In addition, recent evidence demonstrates that methylation of the

IGFBP-3 promoter is one mechanism by which the silencing of IGFBP-3

expression in cancer cells is achieved (41).

Despite promising pre-clinical evidence using IGFBP-3 as a cancer

therapeutic (2-4), controversy remains as to the complex role of IGFBP-3 in

various tumors. IGFBP-3 modulates cellular proliferation with dual actions that

either enhance IGFs or inhibit their actions as well as actions that are idependent

of its binding to IGFs (1). Evidence for this duality has been reported in renal cell

(42,43), lung (44,45), and breast and other cancers (46). Interestingly, an

outcome prediction model for prostate cancer was established utilizing HoxC6

and IGFBP-3 expression, as IGFBP-3 was positively associated with Gleason

score (47). However, recent expression profiling of HoxC6 siRNA transfections


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and HoxC6 overexpression identified IGFBP-3 as a potential proapoptotic

repression target of HoxC6 in prostate cancer (48). Clearly, more work needs to

be done to examine the role of IGFBP-3 in cellular proliferation and apoptosis.

We have recently demonstrated in a prostate cancer model, the

requirement for IGFBP-3 secretion and re-uptake by endocytic pathways

(specifically caveolin- and transferrin receptor-mediated) for apoptosis induced

by transforming growth factor (TGF)β (49). After internalization, IGFBP-3 rapidly

localizes to the nucleus where it interacts with RXRα and other factors (1).

Nuclear import is a nuclear localization signal-dependent process and mediated

by importin-β factor (50).

Our observation that IGFBP-3 translocates Nur77 has several important

implications. First, because IGFBP-3 is a biological signal versus the previously

described chemical apoptosis inducers (i.e. calcium ionophores, etoposide,

rexinoids) (24); this implies that the normal prostate epithelial cell has an

endogenous signal (IGFBP-3), which can induce a programmed cell death

cascade upon cancer surveillance. In fact, we have recently reported that

EWS/FLI-1, an abnormal transcription factor resulting from oncogenic fusion in

Ewing’s tumor, binds the IGFBP-3 promoter in vitro and in vivo and represses its

activity. Moreover, IGFBP-3 silencing can partially rescue the apoptotic phenotype

caused by EWS/FLI-1 inactivation. IGFBP-3-induced Ewing cell apoptosis relies

on both IGF-1-dependent and -independent pathways. These findings therefore

identify the repression of IGFBP-3 as a key event in the development of Ewing's

sarcoma (51).


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IGFBP-3 mediates the effects of multiple anti-proliferative and pro-

apoptotic biological agents including TGFβ (52), tumor necrosis factor-α (53),

retinoids (54), p53 (55), and 1,25 dihydroxyvitamin D3 (56). In addition, IGFBP-3

gene expression is commonly lost in human prostate cancer cell lines and

xenografts, and was detected in DNA microarray analysis of normal compared

with cancerous cells (38). Decreased IGFBP-3 expression is associated with

prostate cancer progression, demonstrating more frequent loss of expression in

advanced disease, in both human and mouse models (37-39). Low IGFBP-3

levels in prostate cancer imply impairment of RXRα/Nur77 translocation and

subsequent apoptosis of cancerous cells.

We have shown that expression of IGFBP-3 and Nur77 together are

additive in their pro-apoptotic effects. The importance of both these genes that

are inactivated on the cellular path to immortalization is supported by the fact that

IGFBP-3 binding and proteolysis is the target of the E7 protein encoded by

human papillomavirus type 15, one of the few viral genes that can immortalize

primary human cells and thereby override cellular senescence (57), and that

Nur77 is inactivated by the Epstein-Barr virus transactivator EBNA2, essential for

the immortalization of B-cells (58). These small, lean, viral genomes would

presumably selectively inactivate critical apoptosis-inducing host proteins that

would hinder the viral program of self-propagation. Additionally, two recent

papers have now described a non-genotropic carrier function of RXRα to

transport Nur77 to the mitochondria to initiate a mitochondria-dependent

apoptotic pathway (36,59).


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Finally, we have described a novel interface between the nuclear receptor

superfamily and the growth and survival-regulating IGF-IGFBP axis. Beyond its

initial description as a serum carrier for the growth-promoting IGFs, IGFBP-3 has

emerged as a multi-functional, intrinsic, IGF-independent, signaling protein that

mediates important autocrine and paracrine regulation of growth and

homeostasis in a variety of tissues (1). Whereas we have previously described

IGFBP-3 binding to the nuclear receptor RXRα and supershifting RXR:RXRE

complexes in EMSA assays, modulating traditional nuclear receptor roles as

transcription factors via modulation of signaling via the RXRE and presumably

taking on a co-activator/co-repressor role in the nucleus (5-7), we currently

describe IGFBP-3 as a modulator of novel nuclear receptor roles as extra-nuclear

mediators of cellular processes. The fact that both IGFBP-3 and Nur77 are

dramatically suppressed by androgens and are upregulated during apoptosis

induced by castration in the ventral rat prostate affords another unique in vivo

model to study IGFBP-3-induced Nur77 translocation (60,61). This phenomenon

also suggests that uncontrolled androgen receptor signalling implicated in

androgen-independent prostate cancer involves the loss of the IGFBP-3 / Nurr77

apoptotic pathway.

It is now well recognized that Nur77 mediates apoptosis (62) through both

transactivation-dependent (63,64) and –independent (17,19,20,22,24) pathways.

The movement of transcription factors (such as RXRα and Nur77), kinases, and

DNA replication factors between the nucleus and cytoplasm is important in

regulating their activity (65). Nur77 interacts with Bcl-2 at the mitochondria,


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inducing a conformational change that exposes its BH3 domain, resulting in Bcl-

2 conversion from an anti- to pro-apoptotic molecule (66). Our results regarding

RXRα/Nur77 suggest a model that may explain how RXRα/Nur77 activity is

regulated, at least partially, by the presence of IGFBP-3. Abnormal Nur77

transcriptional activity may have oncogenic potential because a Nur77 fusion

protein that is 270 times as active as the native receptor in activating gene

expression is produced through chromosomal translocation in extraskeletal

myxoid chondrosarcoma (67). Recent X-ray crystallographic analysis of the

Nur77 Drosophila homologue revealed the absence of both a classic ligand

binding pocket and coactivator binding site (68). Nur77 is often overexpressed in

cancer cells, due to the uncontrolled expression of growth factors that induce its

synthesis and subsequent transactivation (28,61). Agents, such as IGFBP-3, that

induce Nur77 translocation, may inhibit growth and promote apoptosis of cancer

cells.

In conclusion, the elucidation of the signaling pathways involved in the

anti-proliferative, pro-apoptotic effects of IGFBP-3 is important both for our basic

understanding of the mechanism of action of IGFBP-3 on a cellular level and to

devise new therapeutic approaches to treat prostate cancer constituting IGFBP-3

or related derivatives alone or in combination with synergistic agents. Our

present findings indicating that IGFBP-3 induces a rapid RXRα/Nur77

translocation event along with previous findings of modulation of slower DNA

transcriptional events may herald an “amplification” loop in prostate cancer

apoptosis signaling. Mutagenesis and identification of the specific peptide


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regions of each molecule involved in apoptosis signaling will prove to be a

beneficial adjunct to the delivery of these molecules in a broad range of cancer

therapeutics.


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ACKNOWLEDGEMENTS

The authors wish to thank Dr. P. Chambon for use of the F9 RXRα-/- and WT line,

and Dr. D. Mangelsdorf for the GST-RXRα fusion vector. We also thank David

Hwang, John F. Garcia, and Sarah T. Kerfoot for expert technical assistance.

Supported in part, by a Prostate Cancer Foundation award and NIH grants,

RO1AG20954, P50CA92131, RO1CA100938 (PC); as well as a fellowship award

from the Giannini Foundation (KL), a grant from the Stein-Oppenheimer

Foundation (KL), a grant from the Lawson Wilkins Pediatric Endocrinology

Society (KL), and NIH grant 2K12HD34610 (KL, PI ERB McCabe). The authors

declare that they have no competing financial interests.


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Figure Legends

Fig. 1. Rapid Activation of Apoptosis by IGFBP-3. A, Time course apoptosis

induction of Mouse Embryonic Fibroblasts (MEFs) after treatment with 1 µg/ml of

IGFBP-3. Apoptosis induction was quantitated by fluorometric measurement of

activated caspase 3/7. Values are represented as percent of Serum Free. B,

Time course of human glioblastoma line A172 apoptosis induction after

treatment with 1 µg/ml of IGFBP-3. Apoptosis induction was quantitated by

fluorometric measurement of activated caspase 3/7. Values are represented as

percent of Serum Free. C, Time course of human prostate cancer line 22RV1

apoptosis induction after treatment with 1 µg/ml of IGFBP-3. Apoptosis induction

was quantitated by fluorometric measurement of activated caspase 3/7. Values

are represented as percent of Serum Free. ** = p<0.005 relative to Serum Free

conditions.

Fig. 2. IGFBP-3 induces nucleo-mitochondrial translocation of Nur77. A,

Indirect immunofluorescent confocal microscopy of 22RV1 cells after treatment

with IGFBP-3. Nur77 is labeled in red. Nuclei are labeled in blue. Note rapid

appearance of cytoplasmic Nur77. SFM, Serum Free Medium B, Western

immunoblot of subcellular fractions of 22RV1 prostate cancer cells after

treatment with 1mcg/ml IGFBP-3, probed for Nur77. PARP, and Hsp60 are

loading controls and show purity of the nuclear fraction. C, 22RV1 mitochondrial

fraction isolated after treatment with 1mcg/ml IGFBP-3. Membrane was probed

with PARP, PMP70, and Cathepsin S to show purity of the mitochondrial fraction.


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29

Fig. 3. Rapid Mitochondrial Accumulation of Nur77by IGFBP-3 occurs via a type

1 receptor independent mechanism. A, Type 1 IGF receptor disrupted mouse

embryonic fibroblasts (R- MEFs) were treated with IGFBP-3 (1 µg/ml) for the

indicated times and apoptosis was measured in a fragmented DNA/histone

ELISA assay. B, The mitochondrial fraction was analyzed for expression of Nur77

by Western blotting. To demonstrate the purity of the mitochondrial fraction,

expression of mitochondrial-specific protein Hsp60 and nuclear-specific protein

poly(ADP-ribosyl) polymerase (PARP), as well as peroxisomal PMP70 and

lysosomal cathepsin S is shown. A representative of 3 separate experiments is

shown. Values are represented as percent of Serum Free. ** = p<0.005 relative

to Serum Free conditions.

Fig. 4. Additive effects on apoptosis by IGFBP-3 and Nur77. A, Release of

cytoplasmic cytochrome c by IGFBP-3. Values are expressed as fold increase

from baseline derived from densitometric analysis of western immunoblots of

mitochondria-depleted cytoplasmic fractions probed with cytochrome c. Values

were normalized for loading with β-actin. **P<0.005 relative to time 0. B, Caspase

activation post transient transfection of IGFBP-3, Nur77; alone and in

combination. Values are nomalized to β-galactosidase expression to adjust for

transfection efficiency. **P<0.005 relative to vector alone, and also for

combination compared to Nur77 alone. C, Western immunoblot of

overexpressing transiently transfected cells whole cell lysates.


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30

Fig. 5. RXRα is required for IGFBP-3-induced Nur77 translocation. A,

Differential subcellular localization of Nur77 to IGFBP-3 in RXRα+/+ and RXRα-/-

cells. Values are expressed as fold increase from baseline derived from Nur77

densitometric analysis of western immunoblots of nuclear fractions of F9 cells

after treatment with IGFBP-3. Values are normalized to nuclear PARP to adjust

for loading. **P<0.005 relative to no treatment. B, Confocal microscopy of F9 cells

transfected with GFP-Nur77. Note extranuclear appearance of GFP-Nur77 after

treatment with IGFBP-3. C, Caspase activation post transfection of Nur77

expression vector. **P<0.005 relative to control vector alone. D, GST-RXRα pull-

down of 22RV1 cell lysates treated with IGFBP-3. IGFBP-3 enhances the ability of

RXRα and Nur77 to physically associate. CCRF-CEM nuclei was used as a

positive control for Nur77 protein expression E, Overexpression of IGFBP-3

enhances mitochondrial RXRα accumulation. Mitochondrial fraction of 22RV1

cells transiently transfected with IGFBP-3 expression vector. Fraction is

immunoblotted with anti- RXRα.


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Figures

0

50

100

150

Caspase 3/7 activation% of SF

C

0

50

100

150

200

250

SF 1h 6h

A17224h

** **

**


0

50

100

150

200

250

300

SF 2h 6h 24h 48h


*

**

**

MEF

A B

SF 2h 24h6h

200

** ** **

22RV1

Fig. 1. Rapid Activation of Apoptosis by IGFBP-3. A, Time course apoptosisinduction of Mouse Embryonic Fibroblasts (MEFs) after treatment with 1 µg/ml ofIGFBP-3. Apoptosis induction was quantitated by fluorometric measurement ofactivated caspase 3/7. Values are represented as percent of Serum Free. B,Time course of human glioblastoma line A172 apoptosis induction aftertreatment with 1 µg/ml of IGFBP-3. Apoptosis induction was quantitated byfluorometric measurement of activated caspase 3/7. Values are represented aspercent of Serum Free. C, Time course of human prostate cancer line 22RV1apoptosis induction after treatment with 1 µg/ml of IGFBP-3. Apoptosis inductionwas quantitated by fluorometric measurement of activated caspase 3/7. Valuesare represented as percent of Serum Free. ** = p<0.005 relative to Serum Freeconditions.


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32

SFM 15 min

1 hr 2 hr

A Nuclear Cytoplasmic

Time (h)

Nur77

PARP

Mitochondrial

0 0.5 1 3

Hsp60

C

0 0.5 1 3 0 0.5 1 3

Time (h)

Nur77

PARPHsp60

PMP70Cathepsin S

B

Fig. 2. IGFBP-3 induces nucleo-mitochondrial translocation of Nur77. A,Indirect immunofluorescent confocal microscopy of 22RV1 cells after treatmentwith IGFBP-3. Nur77 is labeled in red. Nuclei are labeled in blue. Note rapidappearance of cytoplasmic Nur77. SFM, Serum Free Medium B, Westernimmunoblot of subcellular fractions of 22RV1 prostate cancer cells aftertreatment with 1mcg/ml IGFBP-3, probed for Nur77. PARP, and Hsp60 areloading controls and show purity of the nuclear fraction. C, 22RV1 mitochondrialfraction isolated after treatment with 1mcg/ml IGFBP-3. Membrane was probedwith PARP, PMP70, and Cathepsin S to show purity of the mitochondrial fraction.


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33

Nur77

Hsp60

PARP

+IGFBP-3SF

15 min 60 min 180 min

PMP70

Cathepsin S

Apoptosis ELISA% of SF

-

B

0

50

100

150

****

**

SF 2h 6h 12h

A

Fig. 3. Rapid Mitochondrial Accumulation of Nur77by IGFBP-3 occurs via a type1 receptor independent mechanism. A, Type 1 IGF receptor disrupted mouseembryonic fibroblasts (R- MEFs) were treated with IGFBP-3 (1 µg/ml) for theindicated times and apoptosis was measured in a fragmented DNA/histoneELISA assay. B, The mitochondrial fraction was analyzed for expression of Nur77by Western blotting. To demonstrate the purity of the mitochondrial fraction,expression of mitochondrial-specific protein Hsp60 and nuclear-specific proteinpoly(ADP-ribosyl) polymerase (PARP), as well as peroxisomal PMP70 andlysosomal cathepsin S is shown. A representative of 3 separate experiments isshown. Values are represented as percent of Serum Free. ** = p<0.005 relativeto Serum Free conditions.


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34

2

4

6

Fold Increase from baselineCytoplasmic cytochrome c

**

** **

0 min 30 min 60 min 180 min

Fold Increase from baseline Caspase 3/7 Activation

0.5

1

1.5

2

2.5

3

vector BP3 Nur77 BP3

**

**

+Nur77

A B

**

IGFBP-3

Nur77

vector BP3Nur77 BP3+

Nur77

C

βactin

Fig. 4. Additive effects on apoptosis by IGFBP-3 and Nur77. A, Release ofcytoplasmic cytochrome c by IGFBP-3. Values are expressed as fold increasefrom baseline derived from densitometric analysis of western immunoblots ofmitochondria-depleted cytoplasmic fractions probed with cytochrome c. Valueswere normalized for loading with β-actin. **P<0.005 relative to time 0. B, Caspaseactivation post transient transfection of IGFBP-3, Nur77; alone and incombination. Values are nomalized to β-galactosidase expression to adjust fortransfection efficiency. **P<0.005 relative to vector alone, and also forcombination compared to Nur77 alone. C, Western immunoblot ofoverexpressing transiently transfected cells whole cell lysates.


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35

- + IGFBP-3

IGFBP-3 - + - +

F9WT

F9RXR-/-

3000

4000

5000

6000

Cas

pas

e 3/

7 ac

tiva

tio

nA

rbit

rary

Un

its Control vector

Nur77 vector

F9WT F9RXR-/-

**

F9WT F9RXR-/-

100

200

300

% o

f B

asel

ine

Nu

clea

rN

ur7

7

**

**

Nur77

Cell lysate

IGFBP-3GST-RXRα

CCRF-CEM

MitochondrialRXRα

Control IGFBP-3

+

-

+

- -

+

-

++ +

-

+

-

+

-- - --

+

A B

CD

E

Fig. 5. RXRa is required for IGFBP-3-induced Nur77 translocation. A,Differential subcellular localization of Nur77 to IGFBP-3 in RXRα+/+ and RXRα-/-

cells. Values are expressed as fold increase from baseline derived from Nur77densitometric analysis of western immunoblots of nuclear fractions of F9 cellsafter treatment with IGFBP-3. Values are normalized to nuclear PARP to adjustfor loading. **P<0.005 relative to no treatment. B, Confocal microscopy of F9 cellstransfected with GFP-Nur77. Note extranuclear appearance of GFP-Nur77 aftertreatment with IGFBP-3. C, Caspase activation post transfection of Nur77expression vector. **P<0.005 relative to control vector alone. D, GST-RXRα pull-down of 22RV1 cell lysates treated with IGFBP-3. IGFBP-3 enhances the ability ofRXRα and Nur77 to physically associate. CCRF-CEM nuclei was used as apositive control for Nur77 protein expression E, Overexpression of IGFBP-3enhances mitochondrial RXRα accumulation. Mitochondrial fraction of 22RV1cells transiently transfected with IGFBP-3 expression vector. Fraction isimmunoblotted with anti-RXRα.


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Kuk-Wha Lee, Liqun Ma, Xinmin Yan, Bingrong Liu, Xiao Kun Zhang and Pinchas Cohennucleo-mitochondrial translocation of RXRalpha/Nur77

Rapid apoptosis induction by IGFBP-3 involves an IGF -independent

published online February 24, 2005J. Biol. Chem.

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