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Carcinogenesis Advance Access originally published online on April 13, 2007
Carcinogenesis 2007 28(8):1653-1658; doi:10.1093/carcin/bgm088
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Contribution of the orphan nuclear receptor Nur77 to the apoptotic action of IGFBP-3

Kuk-Wha Lee*, Laura J. Cobb, Vladislava Paharkova-Vatchkova, Bingrong Liu, Jeffrey Milbrandt1 and Pinchas Cohen

Division of Pediatric Endocrinology, Mattel Children’s Hospital at University of California at Los Angeles, David Geffen School of Medicine, 10833 Le Conte Avenue, MDCC 22-315, Los Angeles, CA 90095, USA
1 Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA

* To whom correspondence should be addressed. Tel: +1 310 825 6244; Fax: +1 310 206 5843;Email: kukwhalee{at}mednet.ucla.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Tumor suppression by insulin-like growth factor-binding protein-3 (IGFBP-3) has been demonstrated to occur via insulin-like growth factor-dependent and -independent mechanisms in vitro and in vivo. We have recently described IGFBP-3-induced mitochondrial translocation of the nuclear receptors RXR{alpha}/Nur77 in the induction of prostate cancer (CaP) cell apoptosis. Herein, we demonstrate that IGFBP-3 and Nur77 associate in the cytoplasmic compartment in 22RV1 CaP cells. Nur77 is a major component of IGFBP-3-induced apoptosis as shown by utilizing mouse embryonic fibroblasts (MEFs) derived from Nur77 wild-type and knockout (KO) mice. However, dose–response experiments revealed that a small component of IGFBP-3-induced apoptosis is Nur77 independent. Reintroduction of Nur77 into Nur77 KO MEFs restores full responsiveness to IGFBP-3. IGFBP-3 induces phosphorylation of Jun N-terminal kinase and inhibition of Akt phosphorylation and activity, which have been associated with Nur77 translocation. Finally, IGFBP-3 administration to CaP xenografts on SCID mice induced apoptosis and translocated Nur77 out of the nucleus. Taken together, our results verify an important role for the orphan nuclear receptor Nur77 in the apoptotic actions of IGFBP-3.

Abbreviations: CaP, prostate cancer; IGF, insulin-like growth factor; IGFBP-3, insulin-like growth factor-binding protein-3; JNK, Jun N-terminal kinase; KO, knockout; MEF, mouse embryonic fibroblast; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; WT, wild type


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Prostate cancer (CaP) continues to be the most frequently occurring malignancy (aside from skin cancers) in American men. Large epidemiological studies have shown that serum insulin-like growth factor (IGF)-I levels are elevated and insulin-like growth factor-binding protein-3 (IGFBP-3) levels are reduced in patients with CaP, with differences in these serum markers becoming evident 5 years prior to the development of clinical CaP (1). Multiple lines of in vitro and clinical evidence point to IGFBP-3 as an anticancer molecule (2). Indeed, in vivo demonstration of tumor suppression by IGFBP-3, either administered to cancer xenografts in combination with an RXR ligand (3) or as evidenced by mice transgenic for IGFBP-3 crossed with animal tumor models (4), has been verified by several groups.

We previously reported that IGFBP-3 induces the intrinsic (mitochondria-dependent) pathway of apoptosis by causing the nuclear receptors RXR{alpha}/Nur77 to target mitochondria and induce cytoplasmic cytochrome c release, caspase activation and subsequent apoptosis (5). The orphan nuclear receptor Nur77-dependent apoptotic pathway is unique in that mitochondria-targeted Nur77 induces a conformational change in the pro-survival molecule Bcl-2 and converts it to a ‘killer’ (6), exemplifying a shifting paradigm for transcription factors at the mitochondria (7). Herein, we report the physical interaction of IGFBP-3 and Nur77, the contribution of Nur77 on IGFBP-3-induced apoptosis and demonstrate for the first time that Nur77 translocation by IGFBP-3 occurs in cancer cells in vivo.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 References
 
Materials
Insmed (Glen Allen, VA) provided recombinant human IGFBP-3. Commercial antibodies included the following: anti-human IGFBP-3 from DSL (Webster, TX), anti-Nur77 and blocking peptide were from Geneka Biotechnology (Montreal, Canada) and control goat IgG from Santa Cruz Biotechnologies (Santa Cruz, CA). p-Akt, total Akt, p-Jun N-terminal kinase (JNK) and total JNK antibodies as well as Akt kinase assay were from Cell Signaling Technology (Danvers, MA). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) reagents, Tween and fat-free milk were purchased from Bio-Rad (Hercules, CA). Enhanced chemiluminescence reagents were from Amersham Pharmacia Biotech (Sunnyvale, CA). Full-length IGFBP-3 and Nur77 cDNAs were cloned into pLP-IRESneo mammalian expression vector via pDNR-mediated CreatorTM technology (Clontech, Palo Alto, CA). LipofectAMINE and PLUS reagent were from Invitrogen (Carlsbad, CA). All other chemicals were from Sigma–Aldrich (St. Louis, MO).

Cell culture
22RV1 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Life Technologies, Carlsbad, CA), 100 units of penicillin/ml and 100 units of streptomycin/ml in a humidified environment with 5% CO2. CCRF-CEM cells (American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and 1.0 mM sodium pyruvate, 90%; fetal bovine serum, 10% (Life Technologies), 100 units of penicillin/ml and 100 units of streptomycin/ml in a humidified environment with 5% CO2 also. LAPC-4 cells (8) were a kind gift of Dr C.Sawyers (University of California, Los Angeles) and cultured in Iscove's modified Dulbecco's medium containing 10% fetal bovine serum, 1% L-glutamine and 1 nM R1181 (NEN Life Science Products, Boston, MA).

Subcellular fractionation procedures
Nu-CLEAR Protein Extraction kitTM was from Sigma–Aldrich. Subcellular fractions were isolated according to the manufacturer's protocol.

Transient transfections
Cells (2 x 104) were seeded in 96-well culture plates. Transfections were done with LipofectAMINE:PLUS reagent as directed by the manufacturer (Invitrogen). Typically, 50 ng of ß-galactosidase expression vector (pSV-ß-Gal, Promega, Madison, WI) and 50 ng expression vectors, empty or containing IGFBP-3 and/or Nur77 (to equalize total amount of expression vector), were mixed with carrier DNA to give 0.2 µg total DNA per well. After 24–48 h transfection, caspase activity was quantitated and normalized for transfection efficiency to measurements of aliquots of co-transfected ß-galactosidase gene activity (ß-galactosidase enzyme assay system, Promega).

Co-immunoprecipitation and western immunoblots
22RV1 nuclear or cytoplasmic extracts were immunoprecipitated with or anti-IGFBP-3 antibodies. Briefly, 250 µl of protein A-agarose was incubated overnight at 4°C with 5 µl of anti-human IGFBP-3 antibodies. One hundred and twenty-five microliters of each antibody-treated protein A-agarose were added to 10 µg of protein extract and incubated for 3 h at 4°C with shaking. Immunoprecipitated proteins were pelleted by centrifugation and washed three times with 100 µl of SACI buffer. Sample buffer (200 µl) was added to each sample and vortexed vigorously. Samples were boiled and vortexed again to release protein–antibody complexes from the protein A-agarose. The protein A-agarose was then separated from the immunoprecipitated complexes by centrifugation. The supernatants were saved, and the immunoprecipitated proteins were separated by non-reducing SDS–PAGE (8%) at constant voltage overnight, and then transferred to nitrocellulose for 4 h at 170 mA. The nitrocellulose was immersed in blocking solution [5% non-fat milk/Tris-buffered saline (TBS)] for 45 min, washed with TBS and 0.1% Tween and incubated with primary anti-human Nur77 or anti-human IGFBP-3 antibody (1:4000) for 2 h. After washing off any unbound antibodies, the nitrocellulose was incubated with a secondary antibody (1:10 000) for 1 h. The membrane was washed four times with TBS, 0.1% Tween and TBS. Bands were visualized using the peroxidase-linked enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). Experiments were repeated three times.

Caspase assays
The caspase assay was done using Apo-ONETM homogenous caspase-3/-7 assay (Promega) and performed according to the manufacturer's instructions. rhIGFBP-3 was used at final concentrations of 1–10 µg/ml. Two percent dimethylsulfoxide was used as a control in some experiments.

Generation of Nur77–/– and wild-type primary mouse embryo fibroblast lines
Nur77–/– and wild-type (WT) mice on a C57Bl/6 background were maintained on standard chow. Experiments were conducted in accordance with the Animal Research Committee of the University of California, Los Angeles. Fibroblasts were generated from 13.5-day embryos as described previously (9). Cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1% penicillin–streptomycin and 1% L-glutamine. All cells were used before passage 6.

Kinase assay methods
22RV1 CaP cells were incubated for 24 h in serum-free media in the presence or absence of 1 µg/ml recombinant human IGFBP-3. Akt kinase assay was assessed using a non-radioactive Akt kinase assay kit (Cell Signaling Technology) following the manufacturer's instructions. Briefly, Akt was immunoprecipitated from 100 µg cell lysate overnight using an immobilized Akt antibody. Immunoprecipitated protein was then incubated with 1 µg GSK-3 fusion protein in the presence of ATP for 30 min at 30°C. Proteins were separated by SDS–PAGE and analyzed by autoradiography for phospho and total GSK-3.

Tumor xenografts
The LAPC-4 human CaP cell line expresses a WT androgen receptor and secretes prostate-specific antigen. LAPC-4 xenograft tumors were generated by injection of 1 x 106 cells in 200 µl mixed at a 1:1 dilution with Matrigel in the right flank of male SCID mice. Tumors were established for 2 weeks before the start of treatment. Ten SCID mice with LAPC-4 tumors were treated daily with saline or IGFBP-3 (4 mg/kg/day) given by daily intra-peritoneal injections. The mice were euthanized at 21 days. Tumors were harvested, fixed in formaldehyde and embedded in paraffin.

Immunohistochemical staining for Nur77
Paraffin-embedded, 4 µm thick tissue sections of LAPC-4 xenografts were stained for the Nur77 protein. The sections were deparaffinized in a series of xylene baths and then rehydrated using a graded alcohol series. The sections were then immersed in methanol containing 0.3% hydrogen peroxidase for 20 min to block endogenous peroxidase activity and then incubated in 2.5% blocking serum to reduce non-specific binding. Sections were incubated overnight at 4°C with primary anti-Nur77 antibody (1:100). The sections were then processed using standard avidin–biotin immunohistochemistry techniques according to the manufacturer's recommendations (Vector Laboratories, Burlingame, CA). Diaminobenzidine was used as a chromogen and commercial hematoxylin was used for counterstaining.

TUNEL staining
Paraffin-embedded sections were prepared from LAPC-4 tumors harvested on day 21. After deparaffinization of tissue section, apoptotic DNA fragments were labeled by terminal deoxynucleotidyl transferase and detected by anti-digoxigenin antibody conjugated to fluorescein (ApopTag Fluorescein In Situ Apoptosis Detection kit, Chemicon, Temecula, CA). Cells were examined at x20 using an inverted fluorescence microscope (Axiovert 135M, Carl Zeiss, New York, NY). Apoptotic staining was quantified by pixel histogram (Adobe Systems, Mountain View, CA) and confirmed by manual counting (r = 0.98).

Statistical analysis
All experiments were repeated at least three times. Means ± SDs are shown. Statistical analyses were performed using analysis of variance utilizing InStat (GraphPad, San Diego, CA). Differences were considered statistically significant when P < 0.005, denoted by **.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 References
 
Subcellular association of IGFBP-3 and Nur77
We and others have described the physical interaction between IGFBP-3 and the nuclear receptors RXR{alpha} and RAR{alpha} (10,11). We investigated the ability of IGFBP-3 to associate with Nur77 in nuclear and cytoplasmic compartments utilizing co-immunoprecipitation techniques. 22RV1 CaP cells were taken serum free overnight. Protein A-agarose was used to immunoprecipitate-bound complexes and these were resolved by SDS–PAGE.

Western immunoblotting (Figure 1) showed that endogenous IGFBP-3 associates with Nur77 in cytoplasmic fractions only and complexes were not detected in nuclear fractions. Controls of whole-cell lysate show presence of endogenous Nur77 and IGFBP-3 in the immunoprecipitation input. CCRF-CEM is a T lymphoblastoid cell line (ATCC), and nuclear extract was used as a positive control for Nur77. The major band of endogenous IGFBP-3 was detected in the nucleus, consistent with our previous observations (12). Similarly, Nur77 is typically a nuclear transcription factor. This observation is indicative of distinctive subcellular co-distribution whereby unassociated Nur77 and IGFBP-3 are prevalent in the nucleus, but their complexes are uniquely cytoplasmic.


Figure 1
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  		Fig. 1. Cytoplasmic interaction between IGFBP-3 and Nur77. 22RV1 cells were taken serum free overnight and association of endogenous Nur77 and IGFBP-3 was assessed. Nuclear and cytoplasmic fractions were isolated as indicated. Protein A-agarose was used to immunoprecipitate bound complexes and resolved by SDS–PAGE. Nur77 and IGFBP-3 were detected by immunoblotting. 22RV1 whole-cell lysate (WCL) was used as an input control. CCRF-CEM nuclear extract as a positive control for Nur77 presence. Experiments were repeated three times.

 
Contribution of Nur77 to the apoptotic effects of IGFBP-3
To further study the functional interface of IGFBP-3 and Nur77, we derived Nur77 null (Nur77–/–) embryonic fibroblast cells (MEFs) from the Nur77–/– knockout (KO) mouse. Using these Nur77 null MEFs, we tested the ability of IGFBP-3 to induce apoptosis (Figure 2A). Overnight treatment with IGFBP-3 resulted in a 60% increase in apoptosis as measured by fluorometric measurement of caspase-3/-7 activation in fibroblasts derived from the WT animal, but was not able to induce further caspase activation in the line derived from the Nur77–/–- KO animal. The phenomenon was specific to IGFBP-3-induced apoptosis as caspase activation induced by 2% dimethylsulfoxide was not different in WT versus KO MEFs (Figure 2B). Thus, Nur77 significantly contributes to the pro-apoptotic effect of IGFBP-3 at this dose.


Figure 2
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  		Fig. 2. Nur77 significantly contributes to IGFBP-3-induced apoptosis. (A) Differential response of caspase activation to 2.5 µg/ml IGFBP-3 in MEFs derived from Nur77 WT and KO (Nur77–/–) cells. **P < 0.005 relative to no treatment (Student’s t-test). (B) Specificity of Nur77 involvement for IGFBP-3-induced apoptosis. Note similar responses to dimethylsulfoxide (DMSO) in WT and KO line. (C) Nur77 contributes to IGFBP-3-induced apoptosis in a dose-dependent manner. Dose response to indicated concentrations of IGFBP-3 for 12 h. Caspase-3/-7 activity was measured by fluorometric assay. Experiments were repeated three times.

 
To more closely examine the role of Nur77 in IGFBP-3-induced apoptosis, we treated Nur77 KO and WT MEFs with increasing doses of recombinant IGFBP-3 that ranged from 1 to 10 µg/ml >12 h (Figure 2C). As expected from the former experiment, IGFBP-3 significantly induced apoptosis in WT MEFs in a dose-dependent manner. Surprisingly, IGFBP-3 also significantly induced caspase activation in the KO line in a dose-dependent manner, although this was minimal when compared with the WT line. However, this does suggest that a small portion of IGFBP-3-induced caspase activation may be Nur77 independent, although the functional relevance of this is currently unknown.

Reintroduction of Nur77 in Nur77 KO MEFs restores responsiveness to IGFBP-3
To validate our findings of Nur77 as a mediator of IGFBP-3 action, we reintroduced Nur77 into Nur77 KO MEFs by transient transfection, with and without co-expression of IGFBP-3. Control transfection was with empty expression vectors and was not significantly different from that of transfection with empty Nur77 expression vector and IGFBP-3 over-expression. Over-expression of Nur77 in combination with empty IGFBP-3 expression vector did induce apoptosis activation consistent with previous reports (13,14). Reintroduction of Nur77 via transient transfection restored responsiveness to IGFBP-3 over-expression in the Nur77 KO line (Figure 3). Thus, rescue of IGFBP-3-induced apoptosis was associated with restoration of Nur77 expression.


Figure 3
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  		Fig. 3. Reintroduction of Nur77 into Nur77 KO MEFs restores responsiveness to IGFBP-3. Values are normalized to ß-galactosidase expression to adjust for transfection efficiency. **P < 0.005 relative to control vector alone and also for combination compared with Nur77 alone. Experiments were repeated three times.

 
IGFBP-3 induces phosphorylation of c-JNK and suppression of Akt phosphorylation and activity
Nur77 is phosphorylated by JNK and by Akt (15–17). This phosphorylation is required for its nuclear export and involves JNK activation (phosphorylation) and inhibition of Akt phosphorylation in lung cancer and HEK cells (18). We investigated the effect of IGFBP-3 treatment of cancer cells on JNK phosphorylation and Akt phosphorylation and activation. Treatment with 1 µg/ml of IGFBP-3 for 24 h induced phosphorylation of JNK in 22RV1 CaP cells although the expression levels of total JNK were not changed (Figure 4C). Associated with this is a significant reduction in phosphorylated Akt and Akt activity as evidence by an Akt kinase assay (Figure 4A and B). Thus, the apoptotic action of IGFBP-3, which we have described previously to involved RXR/Nur77 translocation via an IGF-independent mechanism (5), involves inhibition of Akt and phosphorylation of JNK.


Figure 4
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  		Fig. 4. IGFBP-3 induces phosphorylation of c-JNK and suppression of Akt phosphorylation. 22RV1 CaP cells were incubated in serum-free media for 24 h in the presence or absence of 1 µg/ml IGFBP-3. Experiments were repeated three times. (A) Western immunoblot for phospho- and total Akt. (B) Akt kinase assay as assessed by phosphorylation of a GSK-3 fusion protein after immunoprecipitation of Akt. (C) Western immunoblot of phospho- and total JNK. H2O2 was used as a positive control for p-JNK activation. Experiments were repeated three times.

 
IGFBP-3 induces apoptosis and translocates Nur77 in vivo
To determine the effect of IGFBP-3 on Nur77 translocation and apoptosis in vivo, we utilized LAPC-4 cells in Matrigel to create human CaP xenografts on SCID mice. Accordingly, cells were injected and tumors established for 2 weeks. At that time, IGFBP-3 or saline was given as a daily injection at a dose of 4 mg/kg/day intra-peritoneally for 21 days, after which mice were euthanized. Tumor sections were stained with a Nur77 antibody to assess subcellular localization in response to IGFBP-3 as well as subjected to TUNEL staining as a marker for apoptosis. Consistent with our in vitro data, Nur77 exhibited a predominantly nuclear staining pattern in the rapidly growing control tumors (Figure 5A, upper panels). Treatment with IGFBP-3 resulted in predominantly cytoplasmic staining of Nur77 with hematoxylin-counterstained nuclei prominent (Figure 5A, lower panels). Associated with this was a marked significant increase in staining for TUNEL as a marker of apoptosis (Figure 5B). In concert, these data support a novel extranuclear mechanistic role for the orphan receptor Nur77 in mediating the apoptotic actions of IGFBP-3 in vitro and in vivo.


Figure 5
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  		Fig. 5. IGFBP-3-induced apoptosis involves translocation of Nur77 in CaP xenografts in vivo. (A) Cross-sectioned LAPC-4 tumors stained with anti-Nur77 (x100, oil immersion). Diaminobenzidine was used as a chromogen (dark brown) and commercial hematoxylin was used for counterstaining (blue nuclei). Note predominant nuclear Nur77 staining in control tumor versus empty blue nuclei in the IGFBP-3-treated tumor. Top panel is x40 magnification; lower panel is x100 magnification (oil immersion). Arrows indicate nuclei in which Nur77 is intra-nuclearly stained or translocated to the mitochondria. Control immunohistochemistry shows competition of signal by blocking peptide, as well as lack of binding of secondary antibody. (See Supplementary material, available at Carcinogenesis Online, for coloured figure.) (B) Quantitation of TUNEL staining was used as a measure of apoptosis. TUNEL staining was quantitated by pixel histogram as indicated in bar graph. **P < 0.005 relative to control treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 References
 
In the prostate, IGFs and IGFBPs play an important role in the proliferative processes that lead to benign prostatic hyperplasia and CaP. IGFBP-3 is secreted and reuptaken by endocytic pathways (specifically caveolin and transferrin receptor mediated) for apoptosis induced by transforming growth factor-ß (12). After internalization, IGFBP-3 rapidly localizes to the nucleus where it interacts with RXR{alpha}, RAR and potentially RNA Polymerase II binding subunit as recently described (10,11,19), implicating IGFBP-3 as a potential direct modulator of gene transcription. Indeed, IGFBP-3 modulates signaling of nuclear receptor ligands and alters their action (20). Nuclear import of IGFBP-3 is a nuclear localization signal-dependent process and mediated by binding to importin-ß (21).

The first in vivo validation for IGF-independent actions of IGFBP-3 on tumor suppression has recently been published in a mouse model of CaP (4). Important findings in this study include the following: (i) dramatic tumor suppressive effects by a non-IGF-binding mutant of IGFBP-3, (ii) observation that autocrine/paracrine IGFBP-3 expression correlated with tumor suppression, rather than circulating serum levels, and (iii) no obvious effect of IGFBP-3 on non-cancerous tissues. IGFBP-3 transgenic mice have been described to be modestly growth restricted and glucose intolerant, in mechanisms that may be largely related to IGF inhibition (22). However, in vitro evidence would suggest that this too may also involve IGF-independent effects (23). It is probable that growth inhibiting and apoptosis-promoting effects by IGFBP-3 in the whole animal involve IGF-dependent and -independent mechanisms and both are major contributors to tumor suppression.

Although we have demonstrated using the Nur77 KO MEFs that a significant portion of IGFBP-3-induced caspase activation is Nur77 dependent, we were able to show a small but significant contribution that is Nur77 independent. This may be a cell type-specific phenomena or may involve activation of other various pathways that have been described for IGFBP-3 including the following: (i) activation of caspase 8 and 9 (24), (ii) cell-surface receptors (25), (iii) Stat1 activation (26), (iv) SMAD inhibition (27), (v) phosphatase activation (28) and (vi) calcium flux regulation (29).

In addition to IGFBP-3, Nur77-dependent apoptosis is induced by retinoid-related molecules (30,31), calcium ionophores and etoposide (30), which are known to act via signaling pathways that involve kinases and phosphatases. Indirect inactivation of survival kinases by different agents are used to elicit tumor death (32,33). A recent report implicates both activation of JNK and inhibition of Akt as necessary in translocation of Nur77 from the nucleus to the cytoplasm in other cancer models (18). We describe herein JNK activation and inhibition of Akt phosphorylation and activity by IGFBP-3, which translocates RXR/Nur77 in an IGF-independent manner (5). Other pro-apoptotic agents, such as oridonin (34), the multikinase inhibitor, sorafenib, and the proteasome inhibitor, bortezomib (35), have also been described to inhibit Akt and phosphorylate JNK, it is intriguing to speculate that Nur77 translocation may be involved as well.

Additional kinase pathways phosphorylate Nur77 including the mitogen-activated protein kinase pathway (36) and ribosomal S6 kinase and mitogen- and stress-activated kinase (37). We and others have additionally identified DNA protein kinase to phosphorylate IGFBP-3 in vivo and that this phosphorylation regulates nuclear localization (38,39). Additionally, casein kinase-2 has been shown to phosphorylate and negatively regulate IGFBP-3 (40). Complex kinase networks that control phosphorylation of both Nur77 and IGFBP-3 underscore the importance of multiple regulatory levels to determine activity of both these molecules and their subcellular localization.

In summary, the current work and previous work done by our group shows that IGFBP-3 is secreted and reuptaken by endocytic mechanisms to the nucleus (12). In the nucleus it interacts with the nuclear receptor RXR, where binding may expose a nuclear export sequence described previously in the DNA-binding domain of RXR (41) and indirectly enhance Nur77 binding to RXR (5). Conformation of this complex may further be changed after export to the cytoplasm where IGFBP-3 directly interacts with Nur77 as described in the current study. Immunohistochemical demonstration of the ability of IGFBP-3 to translocate Nur77 in vivo offers an output measure to assess IGFBP-3 efficacy in the clinical arena. Further work, including mitochondrial targeting of this complex, is needed to unravel the molecular mechanisms of IGFBP-3 intracellular activity and its potential clinical application.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
References
 
Supplementary figure can be found at http://carcin.oxfordjournals.org/.


   Acknowledgments
 
The authors wish to thank David Hwang and George Lin for expert technical assistance. We are also grateful to Dr X.-k.Zhang for providing the Nur77 coding sequence. This work was supported in part by a CaP Foundation award and National Institutes of Health Grants RO1AG20954, P50CA92131 and RO1CA100938 (to P.C.); grants from the Stein-Oppenheimer Foundation, the Lawson Wilkins Pediatric Endocrinology Society, University of California, Los Angeles, CaP SPORE Career Development Award, National Institutes of Health Grant 2K12HD34610 (to K.-W.L.) and a Department of Defense awards PC050754 (to L.J.C) and PC061077 (to K.-W.L.).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

  1. Chan JM, et al. Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study. Science (1998) 279:563–566.[Abstract/Free Full Text]
  2. Ali O, et al. Epidemiology and biology of insulin-like growth factor binding protein-3 (IGFBP-3) as an anti-cancer molecule. Horm. Metab. Res. (2003) 35:726–733.[CrossRef][Web of Science][Medline]
  3. Liu B, et al. Combination therapy of insulin-like growth factor binding protein-3 and retinoid X receptor ligands synergize on prostate cancer cell apoptosis in vitro and in vivo. Clin. Cancer Res. (2005) 11:4851–4856.[Abstract/Free Full Text]
  4. Silha JV, et al. Insulin-like growth factor binding protein-3 attenuates prostate tumor growth by IGF-dependent and IGF-independent mechanisms. Endocrinology (2006) 147:2112–2121.[Abstract/Free Full Text]
  5. Lee KW, et al. Rapid apoptosis induction by IGFBP-3 involves an insulin-like growth factor-independent nucleomitochondrial translocation of RXRalpha/Nur77. J. Biol. Chem. (2005) 280:16942–16948.[Abstract/Free Full Text]
  6. Lin B, et al. Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3. Cell (2004) 116:527–540.[CrossRef][Web of Science][Medline]
  7. Moll UM, et al. p53 and Nur77/TR3-transcription factors that directly target mitochondria for cell death induction. Oncogene (2006) 25:4725–4743.[CrossRef][Web of Science][Medline]
  8. Craft N, et al. A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase. Nat. Med. (1999) 5:280–285.[CrossRef][Web of Science][Medline]
  9. Sell C, et al. Simian virus 40 large tumor antigen is unable to transform mouse embryonic fibroblasts lacking type 1 insulin-like growth factor receptor. Proc. Natl Acad. Sci. USA (1993) 90:11217–11221.[Abstract/Free Full Text]
  10. Liu B, et al. Direct functional interactions between insulin-like growth factor-binding protein-3 and retinoid X receptor-alpha regulate transcriptional signaling and apoptosis. J. Biol. Chem. (2000) 275:33607–33613.[Abstract/Free Full Text]
  11. Schedlich LJ, et al. Insulin-like growth factor binding protein-3 prevents retinoid receptor heterodimerization: implications for retinoic acid-sensitivity in human breast cancer cells. Biochem. Biophys. Res. Commun. (2004) 314:83–88.[CrossRef][Web of Science][Medline]
  12. Lee KW, et al. Cellular internalization of insulin-like growth factor binding protein-3: distinct endocytic pathways facilitate re-uptake and nuclear localization. J. Biol. Chem. (2004) 279:469–476.[Abstract/Free Full Text]
  13. Weih F, et al. Apoptosis of nur77/N10-transgenic thymocytes involves the Fas/Fas ligand pathway. Proc. Natl Acad. Sci. USA (1996) 93:5533–5538.[Abstract/Free Full Text]
  14. Cheng LE, et al. Functional redundancy of the Nur77 and Nor-1 orphan steroid receptors in T-cell apoptosis. EMBO J. (1997) 16:1865–1875.[CrossRef][Web of Science][Medline]
  15. Kolluri SK, et al. Mitogenic effect of orphan receptor TR3 and its regulation by MEKK1 in lung cancer cells. Mol. Cell. Biol. (2003) 23:8651–8667.[Abstract/Free Full Text]
  16. Masuyama N, et al. Akt inhibits the orphan nuclear receptor Nur77 and T-cell apoptosis. J. Biol. Chem. (2001) 276:32799–32805.[Abstract/Free Full Text]
  17. Pekarsky Y, et al. Akt phosphorylates and regulates the orphan nuclear receptor Nur77. Proc. Natl Acad. Sci. USA (2001) 98:3690–3694.[Abstract/Free Full Text]
  18. Han YH, et al. Regulation of Nur77 nuclear export by c-Jun N-terminal kinase and Akt. Oncogene (2006) 25:2974–2986.[CrossRef][Web of Science][Medline]
  19. Rna polymerase II binding subunit 3 (rpb3), a potential nuclear target of IGFBP-3. Endocrinology (2006) 147:2138–2146.[Abstract/Free Full Text]
  20. Ikezoe T, et al. Insulin-like growth factor binding protein-3 antagonizes the effects of retinoids in myeloid leukemia cells. Blood (2004) 104:237–242.[Abstract/Free Full Text]
  21. Schedlich LJ, et al. Insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-5 share a common nuclear transport pathway in T47D human breast carcinoma cells. J. Biol. Chem. (1998) 273:18347–18352.[Abstract/Free Full Text]
  22. Silha JV, et al. Impaired glucose homeostasis in insulin-like growth factor-binding protein-3-transgenic mice. Am. J. Physiol. Endocrinol. Metab. (2002) 283:E937–E945.[Abstract/Free Full Text]
  23. Chan SS, et al. Insulin-like growth factor binding protein-3 leads to insulin resistance in adipocytes. J. Clin. Endocrinol. Metab. (2005) 90:6588–6595.[Abstract/Free Full Text]
  24. Bhattacharyya N, et al. Nonsecreted insulin-like growth factor binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells by IGF-independent mechanisms without being concentrated in the nucleus. J. Biol. Chem. (2006) 281:24588–24601.[Abstract/Free Full Text]
  25. Huang SS, et al. Cellular growth inhibition by IGFBP-3 and TGF-beta1 requires LRP-1. FASEB J. (2003) 17:2068–2081.[Abstract/Free Full Text]
  26. Spagnoli A, et al. Identification of STAT-1 as a molecular target of insulin-like growth factor binding protein-3 (IGFBP-3) in the process of chondrogenesis. J. Biol. Chem. (2002) 277:18860–18867.[Abstract/Free Full Text]
  27. Fanayan S, et al. Signaling through the Smad pathway by insulin-like growth factor-binding protein-3 in breast cancer cells. Relationship to transforming growth factor-beta 1 signaling. J. Biol. Chem. (2002) 277:7255–7261.[Abstract/Free Full Text]
  28. Ricort JM, et al. Insulin-like growth factor-binding protein-3 activates a phosphotyrosine phosphatase. Effects on the insulin-like growth factor signaling pathway. J. Biol. Chem. (2002) 277:19448–19454.[Abstract/Free Full Text]
  29. Ricort JM, et al. Insulin-like growth factor binding protein-3 increases intracellular calcium concentrations in MCF-7 breast carcinoma cells. FEBS Lett. (2002) 527:293–297.[CrossRef][Web of Science][Medline]
  30. Li H, et al. Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3. Science (2000) 289:1159–1164.[Abstract/Free Full Text]
  31. Dawson MI, et al. Apoptosis induction in cancer cells by a novel analogue of 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid lacking retinoid receptor transcriptional activation activity. Cancer Res. (2001) 61:4723–4730.[Abstract/Free Full Text]
  32. Kawauchi K, et al. Involvement of Akt kinase in the action of STI571 on chronic myelogenous leukemia cells. Blood Cells Mol. Dis. (2003) 31:11–17.[CrossRef][Web of Science][Medline]
  33. Sordella R, et al. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science (2004) 305:1163–1167.[Abstract/Free Full Text]
  34. Jin S. Oridonin induced apoptosis through Akt and MAPKs signaling pathways in human osteosarcoma cells. Cancer Biol. Ther. (2007) 6:261–268.[Web of Science][Medline]
  35. Yu C, et al. Cytotoxic synergy between the multikinase inhibitor sorafenib and the proteasome inhibitor bortezomib in vitro: induction of apoptosis through Akt and c-Jun NH2-terminal kinase pathways. Mol. Cancer Ther. (2006) 5:2378–2387.[Abstract/Free Full Text]
  36. Katagiri Y, et al. Modulation of retinoid signalling through NGF-induced nuclear export of NGFI-B. Nat. Cell Biol. (2000) 2:435–440.[CrossRef][Web of Science][Medline]
  37. Wingate AD, et al. Nur77 is phosphorylated in cells by RSK in response to mitogenic stimulation. Biochem. J. (2006) 393:715–724.[CrossRef][Web of Science][Medline]
  38. Cobb LJ, et al. Phosphorylation by DNA-dependent protein kinase is critical for apoptosis induction by insulin-like growth factor binding protein-3. Cancer Res. (2006) 66:10878–10884.[Abstract/Free Full Text]
  39. Schedlich LJ, et al. Phosphorylation of insulin-like growth factor binding protein-3 by deoxyribonucleic acid-dependent protein kinase reduces ligand binding and enhances nuclear accumulation. Endocrinology (2003) 144:1984–1993.[Abstract/Free Full Text]
  40. Coverley JA, et al. The effect of phosphorylation by casein kinase 2 on the activity of insulin-like growth factor-binding protein-3. Endocrinology (2000) 141:564–570.[Abstract/Free Full Text]
  41. Cao X, et al. Retinoid X receptor regulates Nur77/TR3-dependent apoptosis [corrected] by modulating its nuclear export and mitochondrial targeting. Mol. Cell. Biol. (2004) 24:9705–9725.[Abstract/Free Full Text]
Received September 13, 2006; revised February 28, 2007; accepted March 29, 2007.


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