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Originally published In Press as doi:10.1074/jbc.M111431200 on February 6, 2002

J. Biol. Chem., Vol. 277, Issue 18, 15851-15858, May 3, 2002
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A Zinc-finger Protein, PLAGL2, Induces the Expression of a Proapoptotic Protein Nip3, Leading to Cellular Apoptosis*

Atsushi MizutaniDagger , Takako Furukawa§, Yasushi Adachi, Susumu Ikehara, and Shigeru TaketaniDagger ||

From the Dagger  Department of Biotechnology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan, the § Biomedical Imaging Research Center, Fukui Medical University, Matsuoka-cho, Fukui 910-1193, Japan, and the  First Department of Pathology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan

Received for publication, November 30, 2001, and in revised form, February 4, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pleomorphic adenomas gene-like 2 (PLAGL2) protein containing seven C2H2 zinc finger motifs exhibits DNA binding and transcriptional activation activity and is expressed in response to hypoxia or iron deficiency. To identify the target genes of PLAGL2, we transfected mouse PLAGL2 cDNA into Balb/c3T3 fibroblasts and neuroblastoma Neuro2a cells. Both cells were induced to undergo apoptosis by the expression of PLAGL2 as judged by assays of TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling), DNA fragmentation, propidium iodide staining, and the binding of annexin V to the cell surface. The treatment of the cells with an iron chelator, desferrioxamine, resulted in the induction of apoptosis with a concomitant accumulation of PLAGL2 in the nucleus. The expression of PLAGL2 in Balb/c3T3 cells led to the mRNA expression of a proapoptotic factor, Nip3, which can dimerize with Bcl-2. Nip3 mRNA was also induced in desferrioxamine-treated cells. Furthermore, the Nip3 promoter containing a hypoxia-responsive element was activated by PLAGL2, independent of hypoxia-inducible factor-1 (HIF-1). The transfection of antisense oligonucleotide to mouse Nip3 mRNA into PLAGL2-expressing cells led to a decrease in apoptotic cells compared with sense oligonucleotide-transfected cells. Despite the activation of DNA-HIF-1 binding activity under hypoxic conditions, neither an accumulation of HIF-1alpha nor the activation of HIF-1 was observed following the expression of PLAGL2. These results indicate that PLAGL2 is located downstream of HIF-1 and suggest that PLAGL2 functions as a tumor suppressor in association with HIF-1.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mammalian cells respond to low cellular oxygen tension in part by expressing several genes that encode tissue-specific and ubiquitous proteins (1, 2). These proteins participate in a range of biological processes including erythropoiesis, angiogenesis, glycolysis, and cellular adaptation to stress (1, 2). In solid tumors, low cellular oxygen tension or hypoxia selects for death-resistant cells, which confer a poor prognosis and contribute to cancer progression (3). Under hypoxia, some cells are irreversibly injured and die, whereas others adapt and survive. Although the expression of specific genes within cells seems to be the key to this survival, the factors that determine the fate of individual cells under hypoxia are poorly understood.

Hypoxia-inducible factor-1 (HIF-1),1 a well known mediator of hypoxic response in mammalian cells, is a heterodimer composed of HIF-1alpha and HIF-1beta (4, 5). Although HIF-1beta is expressed constitutively, the expression of HIF-1alpha is precisely regulated by cellular oxygen levels (6). Hypoxia leads to the stabilization of HIF-1 and targeted DNA binding, which initiates the expression of several gene products such as glucose transporters, glycolytic enzymes, erythropoietin, and vascular endothelial growth factor (7, 8).

The PLAG family consisting of PLAG1 and PLAGL1 (also called Zac1) are developmentally regulated C2H2 zinc finger proteins and the main targets for pleomorphic adenomas of the salivary gland (9-11). PLAGL1/Zac1 is a nuclear factor exhibiting DNA binding and transcriptional activation activity and seems to be constitutively expressed in specific murine tissues. Zac1 is also implicated in apoptosis and cell cycle arrest (12). Zac1 was separately isolated in the search for genes whose expression is lost in an in vitro model of cell transformation, hence the name Lot1 for lost on transformation (13, 14). Zac1/Lot1 was found to map to 6q24-q25 (15, 16), a chromosomal region known to harbor a tumor suppressor gene for many types of solid tumors including breast and ovary tumors as well as melanoma (15, 16). The expression in tumor cells demonstrated that Zac1 inhibits tumor cell proliferation through induction of apoptosis and cell cycle arrest (9), but the molecular mechanism behind the antiproliferative activity of Zac1 remains to be clarified.

We recently found that the expression of PLAGL2, another member of the PLAG superfamily exhibiting antiproliferative effects on tumor cells, was induced by hypoxia and iron deficiency (17). PLAGL2 is a ubiquitously expressed nuclear factor, which bound to a GC-rich oligonucleotide whose reporter was transactivated (17, 18). Furthermore, the transient expression of PLAGL2 led to the stimulation of luciferase reporter activities of the LDHA and erythropoietin promoters that carry HRE (17). The promoter activity enhanced in iron-deficient or hypoxic cells was strengthened by PLAGL2 independent of HIF-1.

A considerable portion of the pro-apoptotic versus antiapoptotic cellular proteins of the Bcl-2 family localize to separate subcellular compartments in the absence of a death signal (19) The ratio between pro-apoptotic and antiapoptotic members of this family has been shown to modulate the sensitivity of cells to mitochondrial integrity in response to apoptotic signals. Namely, a substantial fraction of the proapoptotic members localize to cytosol or cytoskeleton prior to the generation of a death signal (19, 20). Activation of the proapoptotic molecule Bax appears to involve translocation and dimerization. In addition, expression of the proapoptotic protein Nip3 in neuroblastoma cells was induced by hypoxia at the transcriptional level, leading to cell death (21). The promoter of the Nip3 gene is responsive to hypoxia and to over-expression of HIF-1alpha via HRE located upstream of the transcriptional start site (21). Thus, HIF-1 has now been shown to play a central role in the hypoxic expression of a variety of genes related to apoptosis and is considered to be a master transcription factor that regulates adaptive gene expression under conditions of oxygen deficiency, but little is known about the role of oxygen-deficient inducible nuclear factors.

Because iron-deficient conditions mimic the effect of oxygen deprivation on a number of hypoxia-responsive genes (17, 21), we mainly studied the role of PLAGL2 by treating cells with an iron chelator, desferrioxamine. Here, we report that the expression of PLAGL2 in the nucleus caused apoptosis mediated by the induction of a proapoptotic protein, Nip3. The treatment of neuroblastoma cells and fibroblasts with desferrioxamine resulted in the induction of apoptosis with indications of PLAGL2 and Nip3. The role of PLAGL2 in the activation of gene expression during apoptosis will be discussed in text.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Transfection-- Mouse Balb/c3T3 fibroblasts and neuroblastoma Neuro2a cells were cultured in Dulbecco's modified Eagle's medium (Nacalai Tesque Co.) supplemented with 10% fetal calf serum (Invitrogen) and antibiotics. To reduce intracellular levels of iron, the cells were incubated in the medium in the presence of 100 µM desferrioxamine for a given period. Balb/c3T3 cells were transfected using a LipofectAMINE reagent (Invitrogen). Neuro2a cells were transfected by electroporation at 960 microfarads (capacitance) and 250 mA as described previously (22).

DNA Probes-- DNA probes were generated by PCR from mouse liver and brain cDNA using the following primers: Nip3, 5'-CATGTCGCAGAGCGGGGA-3' and 5'-CATCAGAAGGTGCTAGT-3'; and Bax, 5'-ATGGACGGGTCCGGGGA-3' and 5'-TCAGCCCATCTTCTTCCA-3'.

Antibodies and Immunoblotting-- Antibodies against mouse PLAGL2 were prepared by injecting a rabbit with 0.5 mg of GST-PLAGL2 (amino acids 252-459) fusion protein in Freund's complete adjuvant. After three subsequent injections at two-week intervals, the resulting antisera were collected and antibodies were affinity-purified using GST-PLAGL2-bound Sepharose CL-4B (23). For immunoblotting, cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore). The conditions for immunoblotting and the detection of cross-reacted antigens were set as described previously (23). The primary antibodies used in this study were anti-HIF-1alpha (StressGen Inc.), anti-actin (Santa-Cruz Biotechnology Inc.), and anti-PLAGL2.

Immunofluorescence Microscopy-- Cells transfected with pCG-PLAGL2 (17) or treated with 100 µM desferrioxamine were incubated at 37 °C for 24 h. They were then washed with PBS (+) (PBS containing 1 mM CaCl2 and 0.5 mM MgCl2), fixed with 4% paraformaldehyde for 20 min, and permeabilized in 0.1% Triton X-100 with PBS (+) for 1 h. After blocking with 2% fetal calf serum in PBS (+), incubation with anti-PLAGL2 as the primary antibody was carried out followed by incubation with Cy3-conjugated goat anti-rabbit immunoglobulin (Amersham Biosciences) (22). To identify apoptotic cells by the binding of annexin V to the cell-surface, the fixed cells were incubated with fluorescein isothiocyanate-labeled annexin V (Molecular Probe) at room temperature for 20 min and then washed three times with PBS (+). The PLAGL2 in the cells and annexin V bound to the cells were visualized by confocal microscopy.

TUNEL Assay-- To detect DNA fragmentation in apoptotic cells by direct end-labeling of cellular genomic DNA with a fluorescein-conjugated dUTP using terminal deoxynucleotidyltransferase enzyme, TUNEL assays were performed using a in situ apoptosis detection kit (Takara Co.).

DNA Ladder Assay-- Cells were collected at 72 h after transfection or after the addition of desferrioxamine. The cells were resuspended in 100 µl of 10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA and were lysed in 100 µl of a lysis buffer (10 mM Tris-HCl, pH 8.0, 20 mM EDTA, 0.5% Triton X-100) at 4 °C (24). After centrifugation at 900 × g for 10 min, the supernatant was withdrawn and treated with phenol/chloroform (1:1, v/v). The cytoplasmic DNA was then precipitated with ethanol (24). The precipitated DNA fraction dissolved in 10 mM Tris-HCl, pH 7.5, containing 1 mM EDTA was treated with 0.1 mg/ml RNase A and 0.1 mg/ml RNase T at 37 °C for 1 h, analyzed on a 2% agarose gel, and detected by ethidium bromide staining.

Estimation of Percentage Apoptosis with Propidium Iodide (PI) Staining-- Cultured cells were harvested using 0.05% trypsin, washed in PBS, and suspended in 70% ethanol at 4 °C for 1 h followed by resuspension in 500 µl of PBS, 250 µl of RNase A (1 mg/ml), and 250 µl of PI (100 µg/ml, Molecular Probes). The percentage of apoptotic cells was measured by the estimation of the G0/G1 subpeak in PI staining using a FACScan (BD PharMingen).

Transfection of Oligonucleotides-- Phosphorothioate sense (5'-ACCATGTCGCAGAGCGGGGA-3') oligonucleotides, which were identical to nucleotide positions 81-100 of mouse Nip3 cDNA (25), and antisense (5'-TCCCCGCTCTGCGACATGGT-3') oligonucleotides were synthesized and purified.

Balb/c3T3 cells (5 × 10 5) transfected with pCG-PLAGL2 were incubated for 16 h and rinsed twice with serum-free Dulbecco's modified Eagle's medium. Phosphorothionate sense or antisense oligonucleotides (10 µM) were transfected into the cells using a LipofectAMINE transfection reagent (Invitrogen). After 4-h transfection, the medium was changed to Dulbecco's modified Eagle's medium containing 7% fetal calf serum and antibiotics. Cells were incubated for an additional 24 h. Cells were collected by using 0.05% trypsin, washed in PBS, and stained with PI.

Northern Blotting-- Total RNA was isolated from Balb/c3T3 and Neuro2a cells using the guanidium isothiocyanate method as described previously (23). The RNA was loaded on a 1% agarose/formaldehyde gel, electrophoresed, and transferred onto a nylon membrane (Amersham Biosciences) for hybridization with 32P-labeled DNA fragments of mouse PLAGL2 cDNA (23) and Nip3 cDNA, and then the filter was hybridized and washed as described previously (23).

Reporter Assay-- Cells were co-transfected with the reporter plasmids, Nip3-Luc and Nip3-Luc(HRE1-Mut) (gifts from Dr. Richard K. Bruick), pSV-beta -galactosidase (Promega), and pCG-PLAGL2 (17) using a LipofectAMINE reagent. The cells were collected at 24 h after the transfection and washed twice with PBS. They were then lysed in a reporter lysis buffer (Promega). The lysate was centrifuged, and the supernatants were assayed for luciferase and beta -galactosidase activities. The luciferase assay was performed according to the protocol for the luciferase assay system (Promega), and the beta -galactosidase assay was performed according to the protocol for the beta -galactosidase assay system (ICN). Transfection efficiency was normalized on the basis of beta -galactosidase activity.

Electrophoretic Mobility Shift Assay-- Nuclear extracts were prepared by the method of Schreiber et al. (26) with some modifications. Balb/c3T3 cells (1 × 107) left untreated or treated with 100 µM desferrioxamine for 8 h were washed with 10 mM Tris-HCl, pH 7.6, containing 0.15 M NaCl and then lysed with 10 mM HEPES, pH 7.9, containing 0.6% Nonidet P-40, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. The homogenates were centrifuged to separate the nuclear fraction from the cytoplasmic fraction. The nuclear pellet was resuspended in 100 µl of 20 mM HEPES, pH 7.9, containing 400 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. The nuclear extracts were obtained by centrifugation at 4 °C. Nuclear extracts were also obtained from Balb/c3T3 and Neuro2a cells cultured under hypoxic conditions (17). The double-stranded oligonucleotide 5'-GCCCTACGTGCTGTCTCA-3' containing the consensus sequence HRE (27) was end-labeled with [gamma -32P]ATP and T4 polynucleotide kinase. Nuclear extracts were incubated with 32P-labeled probes (25,000 cpm) in a reaction buffer containing 25 mM HEPES, pH 7.9, 0.5 mM EDTA, 50 mM KCl, 10% glycerol, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 100 µg/ml poly(dI·dC) with or without the competitor (28). After a 30-min incubation at room temperature, the reaction mixture was loaded onto 4% polyacrylamide gels containing 50 mM Tris, 380 mM glycine, and 1 mM EDTA, pH 8.5, and electrophoresed at 100 V at 4 °C. The gels were exposed to x-ray film at -80 °C.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of PLAGL2 Caused Apoptosis of Mouse Balb/c3T3 Fibroblasts and Neuroblastoma Neuro2a Cells-- Previous studies (9, 12) demonstrated that the expression of Zac1, a homologue of PLAGL2 following transfection, inhibited the proliferation of tumor cells and caused their death. To elucidate whether PLAGL2 triggers apoptosis, PLAGL2 cDNA was transfected into mouse Balb/c3T3 cells and the analyses of TUNEL and DNA ladder assays were carried out. First, to visualize the PLAGL2 accumulated in the cells after transfection of PLAGL2 cDNA, recombinant PLAGL2 was expressed in Escherichia coli and used to obtain antibodies in rabbits. Fig. 1 shows immunoblot analysis of PLAGL2 with PLAGL2 cDNA-transfected Balb/c3T3 cells. The obtained anti-PLAGL2 reacted with a single protein corresponding to the molecular mass of 55 kDa in PLAGL2-expressing cells. PLAGL2 expressed was detected only in the nuclear fraction but not in the cytoplasmic fraction. Excess GST-PLAGL2 fusion protein completely blocked the binding of the antibody to PLAGL2, indicating that the antibody was specific for PLAGL2 protein. Indirect immunofluorescence observations also showed that PLAGL2 protein was found in the nucleus (Fig. 2A), consistent with the nuclear localization reported in COS7 cells expressing hemagglutinin-tagged PLAGL2 (17). As compared with control cells, BALB/c3T3 cells expressing PLAGL2 were clearly TUNEL-positive (Fig. 2A). As expected, the DNA-laddering assay showed DNA to be fragmented in cells expressing PLAGL2 but not in control cells (Fig. 2B). To estimate the G0/G1 subpopulation as apoptotic cells, we performed PI staining, and the proportion of apoptotic cells of PLAGL2 cDNA-transfected Balb/c3T3 cells was analyzed by flow cytometry. As shown in Fig. 2C, the expression of PLAGL2 induced apoptosis, reaching 20.7% of total cells at 48 h after transfection. Early in apoptosis, phosphatidylserine is translocated from the inner to outer leaflet of the plasma membrane. Annexin V, a membrane-impermeable protein, binds phosphatidylserine on intact cells only if phosphatidylserine is present on the outer leaflet (29). The binding of fluorescein isothiocyanate-labeled annexin V to the surface of the apoptotic cells was then examined. The binding was only observed on PLAGL2-expressing Balb/c3T3 cells. When PLAGL2 cDNA was transfected into Neuro2a cells by electroporation, apoptotic cells were also distinguished by the binding of annexin V to the cell surface as compared with the cells transfected with control pCG vector (Fig. 2D). TUNEL-positive Neuro2a and porcine epithelial LLC-PK1 cells were also observed following PLAGL2 cDNA transfection (data not shown). This series of experiments demonstrated that PLAGL2 expression in cells causes annexin V staining, an increase in the G0/G1 subpopulation, and DNA fragmentation, a series of events known to occur in cells undergoing apoptosis.


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  		Fig. 1.   Immunoblot analysis of PLAGL2. Balb/c3T3 cells were transfected with the empty vector pCG (lane 1) or pCG-PLAGL2 (lanes 2-5) and incubated for 48 h. Cell lysates (lanes 1, 2, and 5), the nuclear fraction (lane 3), and the cytoplasmic fraction (lane 4) were analyzed by SDS-polyacrylamide gel followed by immunoblotting using affinity-purified anti-PLAGL2 as the primary antibody. The filter similar to that shown in lane 2 was also incubated with anti-PLAGL2 plus GST-PLAGL2 protein (50 µg of protein/ml) (lane 5).


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  		Fig. 2.   Apoptosis induced by expression of PLAGL2. A, assay of PLAGL2 and TUNEL for detecting apoptotic cells. Mouse Balb/c3T3 cells were transfected with expression vectors for PLAGL2, and after 48 h, they were assayed for DNA fragmentation by TUNEL staining. B, DNA fragmentation. Cells transfected with pCG (lane 1) or with pCG-PLAGL2 (lane 2) were maintained for 72 h. DNA was extracted, subjected to agarose gel (2%) electrophoresis, and visualized by ethidium bromide staining and ultraviolet light trans-illumination. M, lambda -phage DNA-HindIII-digested marker. C, flow cytometric analysis. Cells transfected with pCG or with pCG-PLAGL2 were maintained for 48 h. The cells were collected, and PI staining was performed. Representative patterns of PI staining of Balb/c3T3 cells are shown. The figures show percentage of apoptosis. D, annexin V binding. Balb/c3T3 and Neuro2a cells transfected with empty vector (upper panel) or pCG-PLAGL2 (lower panel) were cultured for 24 h. Cells were fixed and incubated with fluorescein isothiocyanate-labeled annexin V.

Increase in PLAGL2 and Induction of Apoptosis in Balb/c3T3 and Neuro2a Cells Treated with Desferrioxamine-- We (17) previously reported that PLAGL2 mRNA was markedly expressed upon treatment of mouse cells with an iron chelator, desferrioxamine. The treatment of Balb/3T3 cells with 100 µM desferrioxamine resulted in an increase in PLAGL2 protein in the nucleus of most cells (Fig. 3A). Little fluorescence was observed in untreated Balb/c3T3 cells. When Neuro2a cells were treated with 100 µM desferrioxamine for 24 h, an increase in PLAGL2 expression was also observed. PLAGL2 protein in desferrioxamine-treated Neuro2a cells was localized to the nucleus in some cells and to both the cytosol and nucleus in others (Fig. 3A). Immunoblot analysis showed that PLAGL2 corresponding to a molecular mass of 55 kDa in Balb/c3T3 and Neuro2a cells was induced to express by the treatment with desferrioxamine (Fig. 3B). To examine whether the treatment with desferrioxamine induces programmed cell death, we assayed for TUNEL signaling, DNA laddering, PI staining, and annexin V binding. As shown in Fig. 3C, DNA laddering was found in both Balb/c3T3 and Neuro2a cells treated with 100 µM desferrioxamine for 72 h. TUNEL-positive Balb/c3T3 cells were also detected following the treatment with 100 µM desferrioxamine (data not shown). The G0/G1 subpopulation of Balb/c3T3 cells increased to be 9.9 and 14.4% by treatment with 50 and 100 µM desferrioxamine for 48 h, respectively (Fig. 3D). When Neuro2a cells were treated with 50 and 100 µM desferrioxamine for 48 h, apoptotic cells were 25.7 and 30%, respectively. The binding of annexin V to the surface of desferrioxamine-treated Balb/c3T3 and Neuro2a cells (Fig. 3E) was markedly increased. The percentage of these cells was positive for annexin V staining, reaching ~40 and 65%, respectively, after 3 days of treatment. These results suggest that the expression of PLAGL2 plays an important role in the triggering of apoptosis.


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  		Fig. 3.   Induction of apoptosis in Balb/c3T3 and Neuro2a cells treated with desferrioxamine. A, induction of PLAGL2 expression. Balb/c3T3 and Neuro2a cells were left untreated (upper panel) or treated with 100 µM desferrioxamine (lower panel) for 24 h. They were then fixed and incubated with anti-PLAGL2 antibody followed by Cy3-conjugated anti-immunoglobulin. B, immunoblot analysis of PLAGL2 in untreated (-) and desferrioxamine-treated (+) cells. Conditions for cell cultures were similar to those described above, and the cellular proteins were analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting using anti-PLAGL2 (upper panel) and anti-actin (lower panel) as the primary antibodies. C, DNA fragmentation. DNA was extracted from Balb/c3T3 and Neuro2a cells left untreated (lanes 1 and 3) or treated with 100 µM desferrioxamine for 72 h (lanes 2 and 4). DNA analysis and visualization were carried out as described in the legend to Fig. 2. D, PI staining. PI staining was performed with Balb/c3T3 and Neuro2a cells left untreated (upper panel) or treated with 50 (middle panel) and 100 µM desferrioxamine (lower panel) for 48 h. E, annexin V binding. Balb/c3T3 and Neuro2a cells left untreated (upper panel) or cultured with 100 µM desferrioxamine for 24 h (lower panel) were fixed and assayed for the binding of fluorescein isothiocyanate-labeled annexin V to the cell surface. DFO, desferrioxamine.

Effects of PLAGL2 on the Transcription of Proapoptotic Factor Nip3-- Nip3 has been shown to be expressed in a variety of cell and tissue types (21, 30) and was markedly induced in most cells in a HIF-1-dependent manner (21). PLAGL2 stimulated the basal activity of the promoter of the hypoxia-inducible LDHA and erythropoietin genes (17). To elucidate whether PLAGL2 influences the expression of Nip3, we assessed the changes in the Nip3 mRNA level upon the expression of PLAGL2. As shown in Fig. 4A, the Nip3 mRNA in desferrioxamine-treated Balb/c3T3 and Neuro2a cells increased, concomitant with the PLAGL2 mRNA. Notably, the Nip3 mRNA in Balb/3T3 cells remarkably increased upon the expression of PLAGL2. The RNA in PLAGL2-expressing cells appeared to be degraded, implying that the cells were dying. We next examined the promoter activity using a reporter containing a fragment of the Nip3 promoter spanning up to 588 bp upstream from the translation start site and luciferase (Nip3-Luc). The co-transfection of PLAGL2 cDNA and the reporter constructs into Balb/3T3 cells resulted in an increase in luciferase activity by 2-fold as compared with the activity without transfection of PLAGL2 (Fig. 4B). The mutation of the HRE located 234 bp upstream of the Nip3 translation start site (Nip3-Luc(HRE1-Mut)) led to a loss of both hypoxia-mediated and HIF-1alpha -mediated responsiveness (21). PLAGL2 activated Nip3-Luc(HRE1-Mut), similar to Nip3-Luc. The reporter activities of Nip3-Luc and Nip3-Luc(HRE1-Mut) in Neuro2a cells were also increased by the expression of PLAGL2 in a dose-dependent manner (Fig. 4C). The reporter activity of Nip3-Luc increased upon the treatment of Balb/c3T3 and Neuro2a cells with 100 µM desferrioxamine and the activation with Nip3-Luc(HRE1-Mut), corresponding to a quarter the activity with Nip3-Luc under the conditions (data not shown). To examine whether the expression of Nip3 directly regulates the apoptosis, Balb/c3T3 cells expressing PLAGL2 were treated with sense or antisense oligonucleotide to Nip3 mRNA. As shown in Fig. 5, the percentage of apoptotic cells of antisense oligonucleotide-treated cells markedly decreased compared with that of sense oligonucleotide-treated cells. These results indicated that PLAGL2 activates the expression of Nip3 at the transcriptional level, which induces apoptosis.


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  		Fig. 4.   Expression of Nip3 in Balb/c3T3 and Neuro2a cells. A, Northern blots. Balb/c3T3 cells transfected with PLAGL2 cDNA were cultured for 0 (lane 1), 16 (lane 2), and 36 h (lane 3). Balb/c3T3 (lanes 4-6) and Neuro2a (lanes 7-9) cells were treated with 100 µM desferrioxamine for 0 (lanes 4 and 7), 16 (lanes 5 and 8), and 36 h (lanes 6 and 9). Total RNA was collected, analyzed by electrophoresis, transferred onto a membrane, and hybridized with radiolabeled probes specific for Nip3 and PLAGL2. 28 S and 18 S rRNA are shown to indicate the amounts loaded. B, luciferase reporter assay with Balb/c3T3 cells. The cells were transfected with a luciferase reporter plasmid containing the Nip3 promoter (Nip3-Luc) or a luciferase reporter plasmid containing HRE mutated in the Nip3 promoter (Nip3-Luc HRE1-Mut), pSV-beta -galactosidase plasmid, and 0.8 µg of pCG-PLAGL2 or empty vector pCG. The cells were cultured for 16 h. Luciferase activity was measured and normalized to the beta -galactosidase activity. C, reporter assay with Neuro2a cells. Conditions for transfection, cell culture, and the measurement of luciferase activity were set as described above. The data are the average of five independent experiments (Bars, mean ± S.D.).


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  		Fig. 5.   Effect of antisense phosphorothionate oligonucleotide to mouse Nip3 mRNA on induction of apoptosis by the expression of PLAGL2 in Balb/c3T3 cells. Cells transfected with pCG-PLAGL2 or empty vector (upper panel) were incubated for 16 h. The cells were then transfected with sense (10 µM) (middle panel) or antisense oligonucleotide (10 µM) (lower panel) to Nip3 mRNA. The cells were further incubated for 24 h after which the cells were collected and stained with PI.

Effect of Hypoxia, Desferrioxamine, and PLAGL2 cDNA Transfection on the Level of HIF-1alpha and the DNA Binding Activity of HIF-1-- Because hypoxia causes apoptosis via HIF-1-dependent gene expression, we examined the relationship between the expression of PLAGL2 and the activity of HIF-1. The level of HIF-1alpha in desferrioxamine-treated Balb/c3T3 cells increased, concomitant with that of PLAGL2 protein, whereas the transient expression of PLAGL2 cDNA in Balb/c3T3 cells did not change the level of HIF-1alpha (Fig. 6A). The binding of HIF-1 to HRE then was examined by electrophoretic mobility shift assay. In Balb/c3T3 cells, hypoxia as well as desferrioxamine caused the formation of a specific band upon the binding of HIF-1 to an oligonucleotide probe containing HRE (Fig. 6B, lanes 1, 2, and 5). The addition of a 100-fold excess of unlabeled probe diminished the intensity of the band (lane 3). The monoclonal antibody for HIF-1alpha also reduced the binding of HIF-1(lane 4). When Neuro2a cells were treated with 100 µM desferrioxamine for 8 h, an increase in the DNA binding of HIF-1 was also observed (lanes 8 and 9). The expression of PLAGL2 in Balb/c3T3 or Neuro2a cells did not change the DNA binding of HIF-1 at all (lanes 7 and 13). These results strongly suggest that PLAGL2 is able to activate the expression of hypoxia-inducible genes without raising the DNA binding activity of HIF-1.


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  		Fig. 6.   Changes in HIF-1alpha level and HIF-1-DNA binding with expression of PLAGL2. A, the level of HIF-1alpha protein Balb/c3T3 cells were left untreated (lane 1) or treated with 100 µM desferrioxamine (lane 2) for 12 h, collected, and lysed as described earlier. Balb/c3T3 cells were also transfected with empty vector (lane 3) or pCG-PLAGL2 (lane 4) and incubated for 12 h. The cellular proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Immunoblotting was performed using anti-PLAGL2, anti-HIF-1alpha , and anti-actin antibodies. B, electrophoretic mobility shift assay of HIF-1. Balb/c3T3 cells were cultured under conditions of normoxia (NOR) or hypoxia (HYP) for 6 h. Balb/c3T3 and Neuro2a cells were treated with 100 µM desferrioxamine (DFO) for 6 h. The cells were also transfected with empty vector (CON) or pCG-PLAGL2 (PLA), and incubated for 12 h. Nuclear extracts were prepared as described earlier. A reaction mixture containing a radiolabeled HRE probe was prepared with nuclear extract. The positions of a specific protein-DNA complex are indicated by arrows.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study showed that PLAGL2 plays an important role in the induction of apoptosis and directly activates the expression of a proapoptotic factor, Nip3. The transfection of PLAGL2 cDNA into Balb/c3T3 fibroblasts and neuroblastoma Neuro2a cells led to the induction of apoptosis, judging by TUNEL assay, DNA fragmentation, PI staining, and the binding of annexin V to the cell surface. These findings were similar to previous observations (9, 12) that the PLAG members, human and mouse Zac1, caused growth inhibition and cell-cycle arrest of various tumor cells, thus leading to apoptosis. We (17) previously found PLAGL2 mRNA to be induced in iron-depleted cell lines and in response to hypoxia. Iron depletion as well as hypoxia caused apoptosis in most cells. Recent studies (1, 31) have documented a hypoxic accumulation of p53, a potent trigger of apoptotic cell death. Despite these important findings, little is known about the hypoxic regulation of genes that are directly involved in cell death or resistance to cell death. We have shown for the first time that prior to apoptosis, PLAGL2 was accumulated in the nuclei of desferrioxamine-treated BALB/c3T3 and Neuro2a cells and induced the expression of proteins promoting apoptosis.

Mouse PLAGL2 is ubiquitously expressed, whereas human PLAGL2 was shown to be highly expressed in fetal tissues (18). The N-terminal region of mouse PLAGL2 is 70% identical to that of mouse Zac-1, whereas the C-terminal region lacks P and PE repeats and has much less homology (20%) (17). On the other hand, unlike mouse Zac-1, which was found to be highly expressed only in the pituitary gland (9), human Zac1 is widely expressed (12). The rat homologue, Lot1, which is 83% identical to mouse Zac1, lacks the PLE and PMQ repeats in its central region but retains the C-terminal P-, Q-, and L-rich sequences and the P and PE repeats that are missing from human Zac1 (13, 32). Rat Lot1 is expressed in a limited number of tissues in normal rats including ovary, pancreas, testis, and uterus (13). The marked differences in the structure and range of expression of Zac1 among humans, rats, and mice are intriguing but remain unexplained. Because mouse Zac1 interacted with nuclear receptors (32), the tissue-specific expression pattern implies that Zac1 is a modulator of nuclear receptor function. In contrast, a widespread expression would suggest that human Zac1 plays an integral and ubiquitous role in regulating nuclear receptor functions. Among the PLAG family, mouse PLAGL2 was the first member found to be a hypoxia- and iron-deficient inducible protein. Given its structural similarity with Zac1, PLAGL2 may contribute to a novel function of the nuclear receptors under hypoxic conditions.

A marked decrease of rat Lot1 and human Zac1 expression was observed in ovarian cancer cell lines (13, 14). The location of the human Zac1 gene is 6q25a, a chromosomal region that has been implicated in the formation of a number of solid tumors (15, 16). The human PLAG1 gene, which is related to the human PLAGL1/Zac1 gene, is located at 8q12, and two types of tumor-associated chromosomal translocations involving the PLAG1 gene result in the ectopic expression of PLAG1 (11, 33). At present, we cannot identify which human and rat PLAG is a hypoxia-inducible protein or which human disease or tumorigenesis, if any, is caused by mutations of the inducible type of PLAG.

Bruick (21) recently reported that the transcription of the gene encoding Nip3 was significantly induced in response to hypoxia. Nip3 is likely to be a direct target of HIF-1 given the presence of a functional HRE located within its promoter for hypoxia responsiveness. No member of the Bcl-2 family of cell death factors other than Nip3 has been shown to be directly regulated at the transcription level by hypoxia. This study demonstrates that PLAGL2 activates the expression of Nip3 at the transcriptional level. Nip3 contains a BH3 domain and a hydrophobic domain in the C-terminal region that functions in membrane targeting and dimerization (19). Nip3 is able to form heterodimers with the antiapoptotic factors Bcl-2 and BclX-L (19, 21) and then promote a release of cytochrome c from mitochondria followed by the events triggering apoptosis. Based on observations that the antisense oligonucleotide to Nip3 mRNA reduced apoptosis of PLAGL2 cDNA-transfected cells, Nip3 overexpressed upon transfection of PLAGL2 cDNA possibly induces apoptosis directly. Under iron-deficient or hypoxic conditions, HIF-1 and PLAGL2 enhance the expression of a proapoptotic factor Nip3. Because PLAGL2 is induced as a consequence of desferrioxamine-induced apoptosis, the induction of apoptosis by desferrioxamine is unlikely to be mediated directly by the accumulation of PLAGL2. Actually, antisense oligonucleotide to Nip3 mRNA did not affect desferrioxamine-induced apoptosis.2 Besides Nip3, Harakiri that is predicted to be a constitutive active proapoptotic molecule may be up-regulated at the transcriptional level in response to death stimuli (34). Moreover, on the basis of the finding that Bax appears to be transcriptionally responsive to p53 (35), it is considered that a broad set of Bcl-2 members may be transcriptionally regulated. Therefore, PLAGL2 may be involved not only in the expression of typical HIF-1-inducible genes including the erythropoietin and LDHA genes (17) but also in the regulation of the expression of many proapoptotic and antiapoptotic factors.

Mouse PLAGL2 and Zac1 activated the transcription from the promoter containing the consensus binding sequence GGGGGCCCC to a similar extent (12, 18). When fused with the Gal4-DNA binding domain, human PLAGL2 showed a weak activation as compared with human Zac1 (18). Furthermore, mouse PLAGL2 and Zac1 can activate the transcription of the LDHA promoter, which contains HRE (17). The activation profiles of the transient reporter assay showed that PLAGL2 and Zac1 act independent of HRE (17). The reporter activity of the LDHA promoter increased under the conditions of hypoxia and iron deficiency and was further strengthened by the transfection of PLAGL2 cDNA. In the transient transfection experiments, transcription from the Nip3 promoter increased with the expression of PLAGL2 (Fig. 4B). The expression of PLAGL2 increased the basal transcription from both the wild type and mutated Nip3 promoters in Balb/c3T3 and Neuro2a cells. When the cells were transfected with HIF-1alpha cDNA or treated with desferrioxamine, the activity of the Nip3 promoter but not the HRE-mutated Nip3 promoter, Nip3-Luc(HRE1-Mut), increased (21). In this study, a small increase was observed in the reporter activity of Nip3-Luc(HRE1-Mut) as compared with that of Nip3-Luc in desferrioxamine-treated cells, suggesting that PLAGL2 induced by the treatment with desferrioxamine contributes to the activation of the reporter activity, whereas HIF-1 is required for the full activation. Because multiple GC-rich regions were found 5' upstream (-588 to -1 bp) from the translation start site of the Nip3 gene (21), PLAGL2 activates the expression of Nip3 by interacting with a GC-rich region. Similar to the effect of PLAGL2 on the LDHA promoter, the recognition site of PLAGL2 in the Nip3 promoter is different from that of HIF-1, although the expression of PLAGL2 can be regulated by HIF-1. These results suggest that PLAGL2 is located downstream of a HIF-1-dependent apoptotic pathway, which is consistent with the observation that neither the accumulation of HIF-1alpha nor the activation of HIF-1-DNA binding occurred following the expression of PLAGL2. On the other hand, hypoxia and iron deficiency caused an increase in PLAGL2 with a concomitant increase in HIF-1alpha and the DNA binding activity of HIF-1 (Fig. 4).

Hypoxia causes p53-mediated growth arrest and apoptosis (3). p53 transactivates numerous genes capable of causing apoptosis such as the proapoptotic Bcl-2 family and a member of the death receptor family, Killer/DR5 (31, 35). Hypoxic induction of p53 expression requires the concomitant accumulation of HIF-1 alpha , which binds to and stabilizes p53 (4, 36). The interaction of p300/CBP with HIF-1 is also essential for transcriptional activation in response to hypoxia (37). Mouse Zac1 can bind to CBP/p300 as well as nuclear receptors (31). The expression of Zac1 in cancer cell lines inhibited the formation of colonies, similar to that of p53 (9, 31). Furthermore, concurrent expression of p53 and Zac1 in vivo may provide superior protection against neoplastic transformation and tumor progression (12). Given that the transcriptional and physiological functions of PLAGL2 are similar to those of mouse Zac1, the transcriptional machinery of PLAGL2 as well as Zac1 would closely resemble that of HIF-1. Therefore, we propose that PLAGL2 functions as a tumor suppressor in conjunction with p53 to maintain the functions of cells or to induce apoptosis in response to environmental stress.

    ACKNOWLEDGEMENTS

We thank Dr. Richard K. Bruick (Department of Biochemistry, University of Texas Southwestern Medical School) for the gift of Nip3-Luc and Nip3-Luc(HRE1-Mut) and critical comments on the manuscript, and Drs. Y. Sokawa and R. Tokunaga for valuable suggestions.

    FOOTNOTES

* This study was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan and the Yamanouchi Foundation for Research on Metabolic Disorders.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. Tel.: 81-75-724-7789; Fax: 81-75-724-7760; E-mail: taketani@ipc.kit.ac.jp.

Published, JBC Papers in Press, February 6, 2002, DOI 10.1074/jbc.M111431200

2 A. Mizutani., T. Furukawa, and S. Taketani, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: HIF-1, hypoxia-inducible factor-1; PLAGL, pleomorphic adenomas genelike; HRE, hypoxia-responsive element; LDHA, lactate dehydrogenase A; PI, propidium iodide; PBS, phosphate-buffered saline; GST, glutathione S-transferase; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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