Dual roles of Nur77 in selective regulation of apoptosis and cell cycle by TPA and ATRA in gastric cancer cells

Qiao Wu1, Su Liu, Xiao-feng Ye, Zhi-wei Huang and Wen-jin Su

Key Laboratory of the Ministry of Education for Cell Biology, and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian Province, China


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nur77 is an orphan receptor. Although Nur77 affects cell proliferation and apoptosis through its capability of binding to a variety of response elements and regulating their transactivation activities, the intrinsic function of Nur77 is not yet fully understood; in particular, its regulation of apoptosis and proliferation has been characterized as cell type-dependent and agent context-dependent. In this study, Nur77 can be seen to regulate apoptosis via its expression and translocation, rather than its transactivation activity in gastric cancer cells. Nur77 was constitutively expressed in BGC-823 cells. The tetradecanoylphorbol-1,3-acetate (TPA) treatment not only resulted in up-regulation of the Nur77 mRNA level, but also led to translocation of Nur77 protein from the nucleus to the mitochondria, and caused the release of cytochrome c. This TPA-induced translocation of Nur77 was in association with the initiation of apoptosis in gastric cancer cells. Although all-trans retinoic acid (ATRA) could not induce apoptosis in BGC-823 cells due to failure of stimulating Nur77 translocation, expression of Nur77 in the nucleus was required for cell growth inhibition by ATRA. Transfection of antisense Nur77 receptor into BGC-823 cells resulted in resistance of cell growth against ATRA inhibition, and the cells were still arrested in the S phase. Furthermore, the action of Nur77 in TPA-induced apoptosis was mediated through a protein kinase C signaling pathway, while mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways were responsible for the regulation of Nur77 mRNA expression. Taken together, the data revealed the dual functioning mechanisms of Nur77 in gastric cancer cells in response to TPA and ATRA.

Abbreviations: ATRA, all-trans retinoic acid; CAT, chloramphenicol acetyl transferase; CHX, cycloheximide; Cyt c, cytochrome c; DAPI, 4,6-diamidino-2-phenylindole; EGF, epidermal growth factor; LMB, leptomycin B; MAPK, mitogen-activated protein kinase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide; NBRE, Nur77-binding response element; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; RXR, retinoidxreceptor; ßRARE, ß retinoic acid response element; TPA, tetradecanoylphorbol-1,3-acetate.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nuclear receptors comprise a superfamily of structurally related transcriptional factors that control a variety of developmental, physiological and behavioral processes (13). The family includes receptors of hormones and vitamins, as well as a majority of orphan members whose physiological functions are poorly understood (4). Nur77 (also known as NGFI-B (nerve growth factor-induced clone B) and TR3 (57) is an orphan receptor, whose ligand is unknown (8). Just like many early-response genes, Nur77 mRNA expression is induced rapidly by a variety of growth stimuli, including growth factors, phorbol esters, calcium ionophores and some stimuli acting via cyclic AMP-dependent synthesis pathways (6,7,9,10). Thus, Nur77 may function as a nuclear messenger in the signal transduction processes, and along with the products of other early-response genes, represent one of the primary effectors in response to environmental stimuli. Nur77, as a monomer, binds to Nur77-binding response element (NBRE) (11), and also as a heterodimer with RXR (retinoidxreceptor), binds to the ßRARE (ß retinoic acid response element) (12,13). Recently, the authors have reported that RXR-selective retinoids may induce retinoic acid receptor ß (RARß) expression, growth inhibition and apoptosis in breast cancer cells, most probably through their activation of RXR–Nur77 heterodimer that binds to the RARß promoter (14). In addition, the authors also found that Nur77 can heterodimerize with another orphan receptor COUP-TF (chicken ovalbumin upstream promoter transcription factor) to modulate the binding activities of a variety of RAREs and their sensitivity to retinoids in lung cancer cells (15).

A variety of evidence suggests that Nur77 is involved in the regulation of apoptosis in different cells types (1622). Recently, a Nur77 homodimer binding site, NurRE was identified and demonstrated to be activated during T-cell apoptosis (16). A high level of Nur77 protein is present in apoptotic T-cell hybridomas and apoptotic thymocytes, but not in growing T cells or stimulated splenocytes. A dominant negative mutant of Nur77 may protect T-cell hybridomas from activation-induced apoptosis (16,17,23). Further constitutive expression of wild-type Nur77 leads to massive apoptosis, while its dominant negative mutant inhibits the apoptotic process accompanying negative selection of thymocytes in transgenic mice (24). Apoptosis induction in lung cancer cells is mediated through cJun-Nur77 pathway, in which induction of cJun activity is critical for Nur77 expression (20). Therefore, these studies reveal an important role of Nur77 in the regulation of apoptosis.

Although the role of Nur77 as a positive regulator for apoptosis has been recently illustrated, the authors found that in lung cancer cells, Nur77 overexpression is associated with retinoic acid (RA) resistance (15), and may contribute to cell proliferation and neoplastic transformation by blocking the inhibitory effect of RA on cell growth. Consistent with observations in this study, Yin et al. (20) also reported that Nur77 caused a delayed apoptotic process in lung cancer cells, despite a high expression of Nur77 mRNA being rapidly induced by AHPN/CD437. Therefore, these studies, combined with the reports mentioned above, demonstrate the divergent functions of Nur77 in the regulation of cell proliferation and apoptosis. To clarify the regulatory behavior of Nur77 in gastric cancer cells, the characteristic effects of tetradecanoylphorbol-1,3-acetate (TPA) and all-trans retinoic acid (ATRA) on Nur77 were examined. Nur77 was distinctly regulated by TPA and ATRA resulting in different behaviors through different signaling pathways. TPA induced Nur77 mRNA expression and led to Nur77 translocation from the nucleus to the mitochondria, and subsequently caused cytochrome c (Cyt c) release from the mitochondria to the cytosol. These effects were accompanied by the initiation of apoptosis, and were mediated through a protein kinase C (PKC) signaling pathway. Although ATRA did not induce Nur77 mRNA expression and its translocation, Nur77 still functioned in the nucleus through arresting cells in G0/G1 phase to inhibit cell growth. Inhibition of Nur77 mRNA expression by transfection of antisense Nur77 receptor led to BGC-823 cell resistance to ATRA inhibition. Mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways might be responsible for the regulation of Nur77 mRNA expression. Taken together, the data might provide new insight into a novel mechanism of dual roles of Nur77 orphan receptor in gastric cancer cells.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell line and culture condition
The human gastric cancer cell line BGC-823 was purchased from the Institute of Cell Biology, Shanghai. Cells were maintained in RPMI-1640 medium, supplemented with 10% FCS, 1 mmol/l glutamine and 100 U/ml penicillin.

Northern blot analysis
Total RNA was isolated by guanidium thiocyanate followed by centrifugation in CsCl solutions as described previously (14). Twenty micrograms of total RNA were analyzed by eletorphoresis through 1% agarose–formaldehyde gel, and transferred to a nylon filter. The nylon filter was pre-hybridized at 42°C for 4 h and then hybridized overnight with [{alpha}-32P]dATP and [{alpha}-32P]dCTP labeled Nur77 probe at 42°C. The same nylon filter was further hybridized thereafter with [{alpha}-32P]dATP and [{alpha}-32P]dCTP labeled ß-actin probe to quantify the amount of total RNA used. After hybridization, the nylon filter was washed twice with 2x SSC and 0.1% SDS at 60°C and finally washed twice with 1x SSC and 0.1% SDS at 60°C. The washed nylon filter was exposed overnight to X-ray film at –80°C.

Western blot analysis
Nuclear extract was essentially prepared according to the method described previously (25). Fifty micrograms of nuclear extract were electrophoresed on 8% denaturing gel and electroblotted onto a nitrocellulose membrane. The membrane was incubated with anti-Nur77 antibody followed by corresponding secondary antibody. The antibody reactivity was detected with an Amersham ECL kit according to the manufacturer’s instruction. To separate the cytoplasmic, nuclear and mitochondrial fractions, cells were suspended in 500 ml of 10 mM Tris–Cl (pH 7.8), 1% Nonidet P-40, 10 mM mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin and 1 mg/ml aprotinin for 2 min at 0°C, then 500 ml of DDW was added, and the cells were allowed to swell for 2 min. Cells were sheared by 10 passages through a 22 gauge needle. The nuclear fraction was recovered by centrifugation at 400 g for 6 min, and the low-speed supernatant was centrifuged at 100 000 g for 30 min to obtain the mitochondrial fraction (pellet) and the cytosolic fraction (supernatant). The mitochondrial fraction (pellet) was further lysed in the buffer [10 mM Tris–Cl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 5mM EDTA (pH 8.0)].

Transient transfection and CAT activity assay
The construct of reporter gene, NurRE (kindly provided by Dr X.K.Zhang, The Burnham Institute, CA) has been described elsewhere (26). The reporter gene expression vector, together with the ß-galactosidase expression vector (pCH110, Pharmacia) was transiently transfected into BGC-823 cells by calcium phosphate precipitation (15). Transfected cells were treated with various agents as required. The cells were then harvested and lysed, and subjected to assay for ß-galactosidase activity and CAT (chloramphenicol acetyl transferase) activity. All CAT activity was normalized by ß-galactosidase activity. The data shown are the means of three separate experiments.

MTT assay
Cells were seeded at 1000 cells/well in a 96 well plate, and treated with TPA (100 ng/ml, Sigma) or ATRA (10-6 mol/l, Sigma). Medium with TPA or ATRA was changed every other day. One week later, cells were stained with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) for 3–4 h. Reduced MTT crystals were dissolved in dimethyl sulfoxide, and then the plate was scanned under an ELISA reader with a 595 nm filter (27). The data represent the means of two independent experiments.

Stable transfection
The antisense Nur77 expression vector [kindly provided by Dr Uemura (21)] was stably transfected into gastric cancer cells BGC-823 by LipofectamineTM (Gibco BRL) as described previously (28). Briefly, cells were seeded in a six well plate and were ~70% confluent at the time of transfection. Transfections were done by the following procedure: 6 ml CeLLFECTINtm in 1.0 ml standard medium was added to each well containing 1.0 ml of standard medium supplemented with the desired expression vector (20 ng), then screened with 600 mg/ml of G418 (Sigma). The expression of endogenous Nur77 mRNA was determined by northern blotting.

Immunofluorescence analysis
Cells were cultured overnight on cover glass and then treated with various agents as required. After being washed with PBS, cells were fixed in 4% paraformaldehyde. To identify Nur77 protein, cells were incubated first with anti-Nur77 IgG antibody (Santa Cruz), and then reacted with corresponding FITC-conjugated anti-IgG (Pharmingen) as the secondary antibody (26). To show mitochondria, cells were incubated with anti-Hsp60 mouse IgG antibody (Santa Cruz) and then reacted with Texas red-conjugated anti-mouse IgG (Sigma) as secondary antibody (29). To visualize nuclei, cells were stained with propidium iodine (50 mg/ml) containing 100 mg of DNase-free RNase A per milliliter. To indicate Cyt c, cells were incubated with anti-Cyt c IgG antibody (Pharmingen), followed by corresponding Cy5-conjugated anti-IgG (Amersham). Fluorescent images were observed and analyzed with a laser-scanning confocal microscope (Bio-Rad MRC-1024ES).

Apoptosis analysis
For morphological analysis (29), cells were treated with various required agents, trypsinized, washed with PBS, fixed in 3.7% paraformaldehyde and then stained with 50 µg/ml of 4,6-diamidino-2-phenylindole (DAPI, Sigma), in which 100 µg/ml of DNase-free RNase A was added to facilitate the examination of nuclei with a fluorescence microscope. The percentage of apoptotic cells was counted among 1000 cells randomly and shown as the apoptotic index. Detection of cytoplasmic histone-associated DNA fragmentation was done on various agent-treated cells by using the cell death ELISA kit (Boehringer, Mannheim, Germany) as instructed in the manufacturer’s protocol. The results represented the mean values of three independent experiments.

Cell cycle analysis
Cells were trypsinized, collected, fixed by ice-cold 70% ethanol for 1 h, stained for 0.5 h (protecting from light) with 50 mg/ml propidium iodide (Sigma) containing 1.0 mg/ml DNase-free RNase A and subsequently analyzed by a Flow Cytometer. The value represents the results of two independent experiments.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Relationship between induction of Nur77 mRNA expression and cell apoptosis in response to TPA and ATRA
Both TPA and ATRA are usually used as the apoptotic stimuli to induce apoptosis in many types of cancer cell lines (14,26,2934). First, the effects of TPA and ATRA on the induction of apoptosis in gastric cancer cell line, BGC-823 cells, were investigated. In DAPI stained cells, many typical apoptotic cells were seen after TPA treatment. The apoptotic cells became smaller and rounded, and the nuclei became condensed and fragmented with brightly stained chromatin (Figure 1AGo). However, when cells were treated with ATRA, apoptotic cells were much fewer compared with the cells treated with TPA (Figure 1AGo). To determine the relative apoptotic rate in response to TPA and ATRA, the enzyme-linked immunosorbent assay was carried out. Figure 1BGo shows that DNA fragmentation increased in a time-dependent manner, which only happened in TPA-treated cells, not in ATRA-treated cells. The highest DNA fragmentation induced by TPA for 48 h was >10-fold that by both control and ATRA induction groups (Figure 1BGo). The MTT assay showed that both TPA and ATRA could effectively inhibit the growth of BGC-823 cells in a dose-dependent manner, with the highest inhibitory rate amounting to 40.56% (for TPA, 100 ng/ml) and 48.75% (for ATRA, 10-6 mol/l), when cells were treated with these two agents for 1 week (Figure 1CGo). Thus, the data demonstrated that growth inhibition of BGC-823 cell by TPA was due to induction of apoptosis, while that by ATRA was not.








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Fig. 1. Effects of TPA and ATRA on induction of apoptosis and expression of Nur77 mRNA in BGC-823 cells. (A) Morphological analysis of apoptotic cells. Cells were treated with TPA (100 ng/ml) or ATRA (10-6 mol/l) for 24 h, and then stained with DAPI as described in the Materials and methods. Nuclear morphology was visualized with a fluorescence microscope. (B) Analysis of DNA fragmentation. Cells were treated with TPA (100 ng/ml) or ATRA (10-6 mol/l) for the indicated time, DNA fragmentation was analyzed by using a cell death ELISA kit. (C) Effects of TPA and ATRA on cell growth inhibition. Cells were treated with TPA or ATRA at the different concentrations indicated for 1 week, then MTT assay was carried out as described in the Materials and methods. (D) Expression of Nur77 mRNA. RNA was prepared from cells treated with TPA (100 ng/ml) or ATRA (10-6 mol/l) for 24 h, and the mRNA expression level was analyzed by northern blot. 18S and 28S RNA indicates the amount of total RNA used. Induction of Nur77 mRNA by EGF (200 ng/ml, 12 h) was used as a positive control. (E) Expression of Nur77 mRNA in cell clone stably transfected with antisense-Nur77 receptor. The empty vector was also transfected into BGC-823 cells as a comparison. (F) Analysis of apoptotic index. Cells were treated with TPA (100 ng/ml) or ATRA (10-6 mol/l) for 48 h, and then stained with DAPI. Apoptotic cells were counted among 1000 cells randomly. BGC, BGC-823 cells; BGC/Vec, BGC-823 cells transfected empty vector; BGC/aNur, BGC-823 cells transfected antisense-Nur77 vector.

 
Many reports show that Nur77 is involved in the regulation of apoptosis in different cell types (1622). The expression of Nur77 mRNA in response to TPA and ATRA was examined next. Nur77 was constitutively expressed in BGC-823 cells (Figure 1DGo). In contrast to the control, the expression of Nur77 mRNA was significantly up-regulated by TPA, but not affected by ATRA (Figure 1DGo). These results suggested that up-regulation of Nur77 mRNA by TPA might be associated with the induction of apoptosis in gastric cancer cells.

To further verify the possibility that TPA-induced apoptosis is correlated with Nur77 mRNA expression, antisense-Nur77 receptor was stably transfected into BGC-823 cells that normally expressed Nur77 mRNA. Figure 1EGo shows that, in contrast to those in wild-type (BGC) and empty vector-transfected control (BGC/Vec), expression of Nur77 mRNA was almost completely inhibited in antisense Nur77-transfected cells (BGC/aNur). TPA treatment caused apoptosis obviously in BGC-823 and BGC/Vec cells, respectively, but relative low apoptosis occurred in BGC/aNur cells (Figure 1FGo). In the case of ATRA treatment, the apoptotic index in BGC/aNur cells was essentially not changed as compared with that in the untreated ones (Figure 1FGo). Taken together, the data clearly demonstrated that although TPA and ATRA could both inhibit the growth of gastric cancer cells, the intrinsic biological mechanism of the two agents might be quite different with regard to their effects on Nur77 mRNA expression and induction of apoptosis.

Translocation but not transactivation of Nur77 induced by TPA
CAT assay was carried out to clarify whether up-regulation of Nur77 mRNA expression by TPA was involved in Nur77 transactivation. When CAT reporter gene expression vector, which contains a Nur77-binding sequence (NurREh (35), was transiently transfected into BGC-823 cells alone, TPA treatment did not activate, but actually somewhat repressed the transcriptional activity of reporter gene (Figure 2AGo). When exogenous Nur77 expression vector, together with NurRE reporter gene expression vector, was cotransfected into BGC-823 cells, weak inhibition of CAT activity, rather than induction, was also observed in the presence of TPA, as compared with the control (Figure 2AGo). A similar result was seen in the treatment of ATRA (Figure 2AGo). As a positive control, epidermal growth factor (EGF), which also up-regulated Nur77 mRNA expression in BGC-823 cells (Figure 1DGo), significantly enhanced transcriptional activity of reporter gene both in the cases of with and without exogenous Nur77 expression vector (Figure 2AGo). Up-regulation of Nur77 mRNA by EGF, therefore, was correlated to its enhanced transactivation activity, but no such correlation could be detected when stimulated by TPA and ATRA. Thus, the data suggest that Nur77 might function through different mechanisms depending on distinct stimulation of agent context.





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Fig. 2. Nur77 translocated from the nucleus to the cytosol by TPA. (A) Effect of TPA and ATRA on the CAT activity of Nur77. Reporter gene expression vector (100 ng) and ß-galactosidase expression vector (50 ng) were transiently transfected into BGC-823 cells with and without the Nur77 expression vector (25 ng). After transfection, cells were treated with TPA (100 ng/ml) or ATRA (10-6 mol/l) for 24 h, and CAT activity was determined by CAT assay. CAT activity induced by EGF (200 ng/ml) was used as a positive control. (B) Translocation of Nur77 in response to TPA, ATRA and CHX. Cells were treated with TPA (100 ng/ml) or ATRA (10-6 mol/l) for 24 h, then immunostained with anti-Nur77 antibody followed by corresponding FITC-conjugated anti-IgG secondary antibody to show Nur77 protein. Simultaneously, cells were stained with PI to display the nuclei. The fluorescent images were visualized with a laser-scanning confocal microscope. For the effect of CHX on the translocation of Nur77, cells were either treated with CHX (10 mg/ml) for 3 h or pre-treated with CHX for 3 h followed by TPA (C + T) for 24 h. They were then immunostained as mentioned above to show the translocation of Nur77. (C) Effect of TPA on Nur77 protein expression. Cells were treated with TPA (100 ng/ml) for the indicated time. The nuclear and cytosolic fractions were prepared as described in the Materials and methods. Nur77 proteins present in the nucleus and the cytosol were revealed by western blot. A

 
The precise mechanism of Nur77 protein in apoptotic events is less understood, except its expression and transactivation (16,17). It has been proposed that subcellular redistribution of protein contributes to the biological function of protein (26,29,36). To examine whether translocation of Nur77 occurred in response to TPA and ATRA, the immunofluorescent localization of Nur77 was conducted using the corresponding Nur77-specific antibody and observed with a laser-scanning confocal microscope. The result illustrated that in BGC-823 cells, Nur77 was much more abundant in the nucleus than in the cytosol. After treatment of TPA for 24 h, Nur77 translocated almost entirely from the nucleus to the cytoplasm (Figure 2BGo). However, in the case of 24 h ATRA treatment, Nur77 protein remained in the nucleus, and no significant translocation could be detected (Figure 2BGo). To further confirm this observation, cytosolic and nuclear protein fractions were prepared, and Nur77 protein level was examined by western blot. As shown in Figure 2CGo, most of the Nur77 protein-intact cells appeared in the nucleus, and only a trace amount in the cytosol. However, TPA treatment-induced translocation of Nur77 protein from the nucleus to the cytosol was time-dependent (Figure 2CGo). Such redistribution was not induced by ATRA; Nur77 protein still remained in the nucleus no matter how long the cells were treated (data not shown). Therefore, this observation was in accordance with the result of Figure 2BGo, and indicated that translocation of Nur77 protein did happen in BGC-823 cells induced by TPA, but not by ATRA.

To verify the fact that cytosolic Nur77 originated from the nucleus rather than being synthesized in cytoplasm, BGC-823 cells were pre-treated with cycloheximide (CHX) to prevent new protein synthesis in the cytoplasm (37). Laser-scanning confocal observation showed that Nur77 could only be seen in the nucleus when undergoing simply treatment with CHX. However, the translocation of Nur77 from the nucleus to the cytoplasm could be continuously promoted by TPA, even though cytosolic protein synthesis had been inhibited by CHX (Figure 2BGo). As cytosolic protein synthesis having been inhibited by CHX, TPA-induced translocation of Nur77 was convincingly demonstrated.

Translocation of Nur77 into and release of Cyt c from mitochondria
Laser-scanning confocal microscopic observation showed some brighter, punctiform particles clearly appeared in the cytoplasm in the process of Nur77 translocation induced by TPA in BGC-823 cells. Since Hui et al. (26) reported that, in human prostate cancer cells LNCap, Nur77 translocated from the nucleus to the mitochondria in the induction of apoptosis, it implied that those brighter and punctiform particles might be Nur77 protein located in the mitochondria. To examine the precise organelle localization of Nur77 protein, which might be linked to Nur77 function, a mitochondrium-specific protein, heat shock protein Hsp60 (26), was used to illuminate the mitochondria. When TPA-treated cells were immunofluorescently stained with anti-Nur77 antibody and anti-Hsp60 antibody simultaneously, the mitochondria displayed a yellow color (Figure 3AGo, TPA, overlay), resulting from the red color of the mitochondria (Figure 3AGo, TPA, Hsp60) overlapped with the green color of Nur77 protein (Figure 3AGo, TPA, Nur77). This demonstrated that Nur77 protein did translocate into the mitochondria. However, a yellow color did not appear in the mitochondria in ATRA-treated cells (Figure 3AGo, ATRA), because ATRA was incapable of inducing Nur77 translocation (Figure 2BGo, ATRA). Therefore, this unique yellow appearance in TPA-treated sample, not only exactly indicated that TPA stimulated the translocation of Nur77 protein into the mitochondria, but also suggested that this translocation might promote the mitochondria executing its role in apoptosis initiation.





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Fig. 3. Precise localization of Nur77 in the mitochondria and release of Cyt c from the mitochondria. (A) Cells were treated with TPA (100 ng/ml) or ATRA (10-6 mol/l) for 24 h, or pre-treated with LMB (L) (1 ng/ml) for 3 h followed by TPA (T) for 24 h. To show the mitochondria (Hsp60), cells were immunostained with anti-Hsp60 antibody followed by Texas red-conjugated secondary antibody. The immunostaining for Nur77 was the same as described in Figure 2BGo. To display Cyt c (Cyt c), cells were immunostained with anti-Cyt c antibody followed by corresponding Cy5-conjugated secondary antibody. The results were analyzed with a laser-scanning confocal microscope. (B) Effect of TPA on Cyt c release. Cells were treated with TPA (100 ng/ml) for the indicated time, then mitochondrial (mito) and cytosol fractions were prepared as described in the Materials and methods. Expression of Cyt c was determined by western blot. (C) Analysis of apoptotic index. Cells were treated with TPA or/and LMB. The method for calculating apoptotic index was the same as in Figure 1FGo.

 
As TPA stimulated the redistribution of Nur77 protein into the mitochondria (Figure 3AGo) and significantly induced apoptosis in BGC-823 cells (Figure 1A and BGo), it should be taken into account that some mitochondrial protein(s) closely related to apoptosis might become active, as a result of the Nur77 redistribution. Next, the behavior of Cyt c, a specific mitochondrial protein that has a known association with apoptosis once released into the cytosol (3840), was examined. Western blot showed that, during TPA treatment, release of a portion of the Cyt c from the mitochondria into the cytosol was time-dependent (Figure 3BGo). This result was further confirmed by the immunofluorescent analysis with the laser-scanning confocal microscope. As shown in Figure 3AGo, Cyt c completely located in the mitochondria of intact BGC-823 cells (Figure 3AGo, Cont., Cyt c), as compared with the location of the mitochondria shown by Hsp60 antibody staining (Figure 3AGo, Cont., Hsp60). Twenty-four hour TPA treatment had been proven to be sufficient to induce the translocation of Nur77 from the nucleus to the mitochondria (Figure 3AGo, TPA, overlay), and the release of Cyt c was throughout the cytosol (Figure 3AGo, TPA, Cyt c). In comparison, neither Nur77 translocation nor Cyt c release could be induced by ATRA (Figure 3AGo, ATRA). Accordingly, at least, Nur77 entering into and Cyt c released from the mitochondria were two parallel processes induced by TPA, and the latter should act as an important part in the initiation of apoptosis.

Finally, to further inquire into whether translocation of Nur77 is a prerequisite for Cyt c release and the subsequent cell apoptosis stimulated by TPA, the apoptotic index and Cyt c were analyzed in the presence of leptomycin B (LMB), which can non-specifically inhibit protein exported from the nucleus (41,42). As expected, Nur77 translocation was inhibited by LMB and still remained in the nucleus, even in the presence of TPA (Figure 3AGo, L + T, Overlay). The Cyt c was still enclosed in the mitochondria (Figure 3AGo, L + T, Cyt c), and the relevant apoptotic index was only 3.87% (Fig. 3CGo). The data demonstrated that translocation of Nur77 from the nucleus towards the mitochondria is required for Cyt c release and apoptosis induction by TPA in gastric cancer cells.

Nur77 is functioning in growth inhibition by ATRA
Now that Nur77 is required for the induction of apoptosis by TPA, is it also functioning in the BGC-823 cell growth inhibition by ATRA? To probe into this question, MTT assay and flow cytometer assay were done on cells transfected with anti-sense Nur77 receptor to measure their sensitivity to ATRA treatment. The results indicated that the transfected cells (BGC/aNur) became resistant to ATRA treatment, the cell growth inhibitory rate decreased obviously from 42.01 (BGC-823 cells) to 8.63% (transfected cells), even when they had been exposed to 10-6 mol/l ATRA for as long as 1 week (Figure 4Go). In addition, flow cytometric analysis showed that 12 h ATRA treatment caused BGC-823 cells to be significantly arrested at the G0/G1 phase, and the percentage of S-phase cells decreased clearly (Table IGo). In contrast, no apparent change in cell cycle phase distribution could be seen in BGC/aNur cells even with ATRA treatment prolonged to 72 h (Table IGo). Therefore, although Nur77 protein did not redistribute by ATRA stimulation in BGC-823 cells, it could still be functioning through some other mechanism in mediating ATRA-induced growth inhibition.



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Fig. 4. Effect of ATRA on the growth inhibition in BGC-823 cells (BGC) and the cells transfected antisense-Nur77 vector (BGC/aNur). Cells were treated with ATRA (10-6 mol/l) for 1 week, and then MTT assay described in the Materials and methods was performed. B

 

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Table I. Cell cycle distribution (%) of gastric cancer cell lines
 
Distinct signaling pathways mediate different effects of Nur77
The target of TPA is PKC (43,44). Thus, it could be inferred that Nur77 might be brought into action through the PKC pathway. Therefore, an examination was carried out on the possible role of PKC pathway in mediating the expression and tranlocation of Nur77. Some PKC-specific inhibitors, such as wortmannin (45) and PKC inhibitor peptide (46), were used to treat cells before TPA and ATRA stimulation. Northern blot showed that the Nur77 mRNA level was markedly enhanced by TPA, but significantly inhibited by either wortmannin (Wort) or PKC inhibitor peptide (PIP) even in the presence of TPA (Figure 5AGo). Another result, evident from laser-scanning confocal microscope observation, indicated that the TPA-stimulated translocation of Nur77 protein was essentially prevented by wortmannin (W) or PKC inhibitor peptide (I) pre-treatment (Figure 5BGo, W + T, I + T). All these results suggested that Nur77 transcription and translocation activated by TPA was mediated through the PKC pathway, and this pathway might play a major role in apoptosis promoted by Nur77.





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Fig. 5. Effects of some specific inhibitors on Nur77. (A) Repression of Nur77 mRNA expression by wortmannin and PKC inhibitor peptide. Cells were treated with either wortmannin (Wort, 100 nmol/l) or PKC inhibitor peptide (PIP, 12 mmol/l), or else pre-treated with these inhibitors for 2 h followed by TPA (100 ng/ml) treatment for 24 h. The total RNA was then prepared. Expression of Nur77 mRNA was detected by northern blot. 18S and 28S were used to quantify the amount of RNA used in each lane. (B) Effects of wortmannin or PKC inhibitor peptide on the translocation of Nur77. Cells were pre-treated with wortmannin (W, 100 nmol/l) or PKC inhibitor peptide (I, 12 mmol/l) for 2 h followed by TPA (T, 100 ng/ml) treatment for 24 h, and then stained with the corresponding antibody similar to Figure 2BGo. (C) Inhibition of Nur77 mRNA expression by PD98059 and LY294002. Cells were either treated with PD98059 (PD, 50 µmol/l) or LY294002 (LY, 50 µmol/l), or else pre-treated with these two inhibitors for 2 h followed by ATRA (10-6 mol/l) treatment for 24 h. Expression of Nur77 mRNA was detected by northern blot. 18S and 28S were used to quantify the amount of RNA used in each lane.

 
In the other experiment, results showed that ATRA rendered BGC-823 cell arrest in G0/G1 phase through MAPK and PI3K signaling pathways (unpublished data). This finding provides the possibility to define the signaling pathway mediating ATRA-stimulated Nur77 function. Figure 5CGo shows that, PD98059, an inhibitor of Raf/ERK, which is a member of the MAPK pathway (47), abolished Nur77 mRNA expression either in the absence or presence of ATRA (Figure 5CGo). A similar result was also seen when LY294002, a PI3K pathway-specific inhibitor (48), was used (Figure 5CGo). Thus, the data suggested that MAPK and PI3K pathways might be intrinsic for the regulation of Nur77 expression in gastric cancer cells.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nur77 orphan receptor functions in the nucleus as a transcriptional factor to positively or negatively regulate gene expression (1215). It has been well known that Nur77, through its capability of binding to a variety of response elements, can regulate its transactivation activity and mediate diverse signaling to affect cell proliferation and apoptosis (14,15,23,49).

Nur77 is constitutively expressed and Nur77 mRNA expression may significantly be up regulated by TPA treatment in BGC-823 cells (Figure 1DGo). TPA can induce apoptosis (Figure 1A and BGo), and ATRA can induce cell cycle arrest (Table IGo) in BGC-823 cell, but both of these effects may be eliminated by antisence Nur77 receptor transfection (Figures 1E and F and 4GoGo, Table IGo), indicating that Nur77 acts as an intrinsic part in these two distinct processes. As the apoptotic effect of Nur77 has been suggested through its transactivation activity in the regulation of gene expression (1619), transactivation activity of Nur77 should be activated during apoptosis induction. However, in BGC-823 gastric cancer cells, transactivation activity of Nur77 was weakly repressed, rather than enhanced, in the TPA-induced apoptotic process, irrespective of the presence or absence of Nur77 expression vector (Figure 2AGo). In addition, in the case of ATRA-induced growth inhibition on BGC-823 cells, Nur77 transactivation activity also showed the similar pattern as that seen in TPA-induced apoptosis (Figure 2AGo). These findings demonstrate that, at least in BGC-823 cell model, although Nur77 does play an intrinsic part in the course of apoptosis- and cell cycle arrest-induction, the way by which it operates in these processes should be other than its transactivation activity.

It has been reported that redistribution of transcription factors, kinases and replication factors between the nucleus and the cytoplasm is critical for the regulation of their activities and the execution of their functions, and phosphorylation is necessary for such redistribution of some proteins (5052). In this study, the results of laser-scanning microscopic observation and western blot analysis indicated that Nur77 actually translocated from the nucleus to the cytoplasm (precisely, into the mitochondria) in response to TPA stimulation (Figures 2B and 2C and 3AGoGo). In addition, the significant apoptosis of BGC-823 cells induced by TPA (Figure 1A and BGo) could be abolished when translocation of Nur77 was blocked by relevant inhibitor, LMB (Figure 3A and CGo). The data, therefore, revealed a novel linkage between TPA-induced apoptosis and translocation of Nur77 in gastric cancer cells. Thus, It was further demonstrated that, in BGC-823 cell model, the mechanism of Nur77 in apoptosis induction relies on its translocation, rather than its transactivation activity.

The data showed that the translocation of Nur77 from the nucleus to the cytoplasm was irreversible (Figure 2BGo, CHX, C + T). It has been documented that the phosphorylation of many members in thyroid/steroid receptor family occurs on serine and threonine (53) or tyrosine (54) residues. Nur77 protein is very rich in serine and threonine and contains numerous possible phosphorylation sites (55). The fact that Nur77 protein in TPA-treated BGC-823 cells exhibited an increased mobility after being incubated with alkaline phosphatase, and this shift in mobility could clearly be blocked by the addition of phosphatase inhibitors, such as NaF, Na3VO4 and Na4P2O7 (unpublished data), indicated that the phosphorylation of Nur77 did occur in the course of TPA induction. Thus, it is probable that phosphorylation of Nur77 is capable of preventing cytoplasmic Nur77 from re-entering the nucleus in gastric cancer cells.

It is interesting to know how Nur77 functions when translocated to the cytoplasm. The precise translocation of Nur77 into the mitochondria (Figure 3AGo) and the resultant release of Cyt c (Figure 3A and BGo) assisted in clarifying the functioning mechanism of Nur77. Cyt c locates at the intermembrane space and on the surface of the inner mitochondrial membrane (56), and its release from the mitochondria plays a critical role in inducing apoptosis (57). Addition of Cyt c and dTPA to cytosolic preparation from growing cells activates caspases, such as CPP32 (58), which is responsible for cleavage of PARA and PKC{delta} (5961). Cyt c also induces DNA fragmentation in isolated nuclei incubated with cytosolic lysates (58). These findings, in association with present results, give rise to the suggestion that translocation of Nur77 to the mitochondria in response to TPA might promote the release of Cyt c into the cytosol and subsequently initiate apoptosis. It can also explain why ATRA could not induce apoptosis in BGC-823 cells (Figure 1A and BGo): this is in accordance with the fact that neither translocation of Nur77 nor release of Cyt c could be induced by ATRA (Figure 2B and 3AGoGo). However, the un-translocated nuclear Nur77 still has its function in mediating ATRA-induced growth inhibition in this cell line, probably through its regulation on some genes (such as nuclear transcription factors, NK-{kappa}B, c-jun and c-myc). These different biological activities of Nur77 strongly suggest that Nur77 may execute dual functions in gastric cancer cells, which is closely related to the subcellular location of Nur77.

Previous works by the authors showed that, in breast cancer cells, heterodimer of Nur77 and RXR binds to RARß promoter, and inhibits the expression of RARß, which is critical in apoptosis induction (14). The same research also noted that, in lung cancer cells, Nur77 heterodimerizes with COUP-TF, blocks the binding of COUP-TF to RARE, and decreases the lung cancer cell’s sensitivity to ATRA (15). Along with the role of un-translocated Nur77 in mediating ATRA-induced growth inhibition in BGC-823 cells mentioned above, it is believable that Nur77 also has its functional activity in nucleus, probably through its interaction with some nuclear components, such as nuclear transcription factors, NF-{Phi}B, c-jun and c-myc, and acts its part in anti-mitogenic or death-inducing effects in different cell types (6264). Of course, further study is required to address the mechanism by which Nur77 interacts with other nuclear components.

The signaling pathway for the mediating Nur77 role is less understood. Pre-treatment with specific inhibitors of PKC, wortmannin or PKC inhibitor peptide, results in the inhibition on both mRNA induction and translocation of Nur77 in BGC-823 cells even in the presence of TPA (Figure 5A and BGo), indicating that the PKC pathway may be involved in TPA-induced and Nur77-mediated apoptosis in gastric cancer cells. This observation may provide a novel mechanism for cross talk between the Nur77 orphan receptor and PKC-signaling pathway. In the authors’ previous study, PKC{alpha} was found to be critical for TPA to induce apoptosis in gastric cancer cells (29). Notably, PKC{alpha} translocated from the mitochondria to the nucleus in response to TPA during the apoptotic process (29). Thus, all of these findings raise the hypothesis that Nur77 and PKCs might interact somewhere (most likely in the mitochondria) during the apoptotic event induced by TPA. Considering some of the available data obtained, it might be in such a case that TPA-induced translocation of Nur77 to the mitochondria causes Cyt c release (Figure 3AGo), which regulates caspase 3 and other proteins relating to apoptosis (unpublished data), then promotes PKCs leaving the mitochondria (29). Certainly, the exact mechanism for this cross talk among Nur77, apoptosis-related protein, and PKCs is yet unknown, and should be further investigated and clarified.

Suppression of Nur77 expression by transfection of antisense Nur77 receptor into BGC-823 cells results in the incapability of ATRA to inhibit BGC-823 cell growth and the cells still arrested in S phase (Table IGo). However, the signaling pathway in relation to such effect of ATRA has not yet been reported. Recent research indicates that intracellular localization of Nur77 is regulated in part by phosphorylation, and this phosphorylation induced by NGF is through the MAPK pathway (64). In addition, Nur77 is a target molecule in the PI3K-dependent AkT pathway: AKT suppresses Nur77-induced apoptosis in fibroblasts and activation-induced cell death of T-cell hybridomas (65). These studies imply that MAPK pathway and PI3K pathway may be involved in Nur77 behavior. In fact, the authors also found that the G0/G1 arrest of BGC-823 induced by ATRA was associated with MAPK and PI3K signaling pathways, and ATRA inhibited AKT expression (unpublished data). In this present study, the fact that MAPK inhibitor PD98059 and PI3K inhibitor LY294002 specifically repressed Nur77 mRNA expression (Figure 5CGo) revealed the possible signaling pathways for Nur77 mRNA expression in gastric cancer cells. So it is probable that in the presence of ATRA, the un-translocated nuclear Nur77 may function as an anti-mitogenic factor through its modulation on the expression of some related proteins in nucleus, such as AKT that was repressed and translocated from the cytoplasm to the nucleus by ATRA in BGC-823 cells (unpublished data).

In summary, Nur77 in BGC-823 cells is essential for TPA and ATRA to exert their effects on gastric cancer cells. Translocation of Nur77 from the nucleus to the mitochondria and the subsequent Cyt c release from the mitochondria to the cytosol are required for TPA to induce apoptosis, this process is mediated through PKC signaling pathway. ATRA does not induce apoptosis in BGC-823 cells in accordance with its failure of inducing translocation of Nur77. However, Nur77 still exerts its function of cell growth inhibition in the nucleus for the cell cycle regulation. MAPK and PI3K signaling pathways might be responsible for regulation of Nur77 expression. Nur77 effects on regulation of apoptosis and cell cycle are coordinative, which finally causes growth inhibition of gastric cancer cells. The present study reveals the novel dual functional roles of Nur77 in gastric cancer cells in response to different stimuli.


    Notes
 
1 To whom correspondence should be addressed Email: xgwu{at}xmu.edu.cn Back


    Acknowledgments
 
Supported by the National Outstanding Youth Science Foundation of China (No. 39825502), the National Natural Science Foundation of China (No. 39880015, 30170477) and the National Natural Science Foundation of Fujian Province (C0110004).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 26, 2002; revised June 6, 2002; accepted June 7, 2002.