Negative Cross-Talk between Nur77 and Small Heterodimer Partner and Its Role in Apoptotic Cell Death of Hepatoma Cells

Myeong Goo Yeo, Young-Gun Yoo, Hueng-Sik Choi, Youngmi Kim Pak and Mi-Ock Lee

College of Pharmacy (M.G.Y., Y.-G.Y., M.-O.L.), Seoul National University, Seoul 151-742, Korea; Hormone Research Center (H.-S.C.), School of Biological Sciences and Technology, Chonnam National University, Kwangju 138-736, Korea; and Asan Institute for Life Science (Y.K.P.), University of Ulsan College of Medicine, Seoul 500-757, Korea

Address all correspondence and requests for reprints to: Mi-Ock Lee, Ph.D., College of Pharmacy, Seoul National University, San 56-1 Sillim, Kwanak, Seoul 151-742, Korea. E-mail: molee{at}snu.ac.kr.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nur77, an orphan nuclear receptor, has been implicated in apoptosis of a variety of cell types, including hepatocytes. The small heterodimer partner (SHP) binds and inhibits the function of many nuclear receptors. Here, we investigated cross-talk between Nur77 and SHP during anti-Fas antibody (CH11)-mediated apoptosis of hepatic cells. Expression of SHP decreased, whereas antisense SHP enhanced, the transcriptional activity of Nur77 in HepG2 cells. SHP and Nur77 were physically associated in vivo and colocalized in the nucleus. SHP decreased the transactivation function of the N-terminal domain of Nur77 that recruits coactivators. Nur77 and SHP competitively bound to cAMP response element-binding protein-binding protein and the expression of coactivators, such as cAMP response element-binding protein-binding protein and activating signal cointegrator-2, recovered the decreased function of Nur77 caused by SHP. Finally, SHP was differentially expressed in hepatoma cell lines in that it was not detected in the interferon-{gamma} (IFN{gamma})/CH11-sensitive SNU354, whereas it was significantly expressed in the IFN{gamma}/CH11-resistant HepG2. Interestingly, a stable SNU354 cell line that expressed SHP became resistant to the IFN{gamma}/CH11-induced apoptosis. Together, our results suggest that SHP plays a key role in the regulation of Nur77 activation and thereby in Nur77-mediated apoptosis in the liver.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUR77 [ALSO KNOWN as nuclear growth factor I-B, N10, TIS1, and Nak-1; nuclear receptor (NR)4A1] is an orphan member of the steroid/thyroid receptor superfamily of transcriptional factors that regulates gene expression (1, 2). Nur77 is composed of an N-terminal transactivation domain, a DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD) (3). The N-terminal region of Nur77 is implicated in the control of subcellular localization, activation function (AF)-1 activity recruiting coactivators, and modulation by kinases such as ERK 2 and Jun N-terminal kinase (4, 5, 6, 7, 8). The mutant lacking the N-terminal transactivation domain acts as a dominant-negative mutant that prevents apoptosis in T cell receptor-stimulated T cell hybridomas (9, 10). Nur77 is called an orphan receptor because the endogenous signaling molecules that bind to Nur77 are not known. Nur77 is constitutively active when overexpressed, suggesting that the orphan receptor does not require ligand stimulation. Recently, it has been reported that 6-mercaptopurin (6-MP), a purine antimetabolite, regulates transactivation of the Nur77 gene family through the activation function (AF)-1 domain (11). Nur77, together with two other NRs, Nurr1 (Not 1) and Nor-1 (MINOR), belongs to the NR4A NR superfamily (3, 12, 13). The subfamily members share extensive homology in DBD and LBD, but diverge in the N-terminal AF-1 domain (3). They bind to the same response elements in promoters, although differential tissue expression and independent roles have also been described for each member.

Nur77 is involved in apoptosis of many cell types in response to a variety of stimuli. Nur77 is rapidly induced by T cell receptor signaling in immature thymocytes and T cell hybridomas, followed by apoptotic cell death (9, 10, 14). Nur77 is involved in activation-induced cell death in macrophages, because Nur77 induction correlates well with cell death, and the cell death is reduced in Nur77-deficient macrophages (15). We and others have shown that Nur77 is important in apoptosis of malignant cancer cells of colon, stomach, and prostate when treated with chemotherapeutic agents (16, 17). Nur77 is up-regulated in the presence of hepatitis B virus (HBV) X protein, which may have a key role in the expression of Fas ligand (FasL) in the hepatic inflammatory lesion caused by HBV infection (18, 19). However, the mechanism by which Nur77 induces apoptosis remains unclear. In some cell lines, Nur77 acts through a transcription-independent mechanism involving translocation to mitochondria, leading to cytochrome c release (20). Recently, it was reported that the interaction of Nur77 with the Bcl-2 apoptotic machinery converts Bcl-2 from a protector to a killer (21). In contrast, the transcriptional function of Nur77 was required to induce many genes, including FasL, TNF-related apoptosis inducing ligand (TRAIL), Nur77 downsream gene (NDG), and NDG2, which activated known and novel apoptotic pathways in thymocytes that carried the Nur77 transgene (22).

The small heterodimer partner (SHP, NR0B2) is an atypical orphan NR that comprises only a putative LBD without a conventional DBD (23). SHP interacts and represses the transcriptional activity of various NRs through several different mechanisms (23, 24, 25, 26, 27, 28, 29, 30). It inhibits the DNA binding of some NRs, including the retinoic acid receptor-retinoic X receptor heterodimer (23). SHP directly binds to estrogen receptors via an LXXLL-related motif in the AF-2 region of the receptor, thereby competing with coactivators that usually bind the motif for the ligand-dependent transactivation (24). It recruits unknown corepressors through its transrepression domain (24, 25, 26). Indeed, it contains a transcriptional repressor domain which recruits E1D1-antagonizing coactivators’ function, thereby contributing to transcriptional inhibitory function (27). SHP seems to repress transcriptional activity of liver X receptor by competing with coactivator, transcriptional intermediary factor 2 (TIF2) (28). SHP interacts with glucocorticoid receptor (GR) through the second NR-box and antagonizes the activity of GR by competing coactivator peroxisomal proliferator-activated receptor-{gamma} coactivator 1 (PGC-1) (29). SHP interacts with the pregnane X receptor (PXR) and repressed its transcriptional activity by both inhibiting DNA binding and squelching steroid receptor coactivator 1 (SRC-1) (30). As a consequence, the physiological role of SHP has been implicated in many aspects of metabolic processes such as bile acid synthesis and glucose homeostasis.

Acute liver failure due to hepatic apoptosis is a symptom of different kinds of liver diseases, such as infection with viral hepatitis, alcoholic hepatitis, and autoimmune hepatitis. Because SHP is abundantly expressed in the liver, cross-talk between SHP and NRs that are rich in the tissue may be involved in apoptosis of hepatocytes in acute and chronic inflammatory liver diseases. In the present study, we examined the potential cross-talk between SHP and Nur77, an important mediator of hepatic apoptosis, and associated molecular mechanisms. Further, we showed that protective function of SHP in the apoptotic death of liver cells which was mediated by Nur77. Taken together, we suggest that SHP functions to regulate Nur77 signaling and play a protective role in the Nur77-mediated apoptosis in liver.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcriptional Activity of Nur77 Is Repressed by SHP
The critical role of Nur77 in Fas/FasL pathway-mediated apoptosis has been well documented in thymocytes (3, 9, 10, 14). Similarly, Nur77 was induced in hepatic cells expressing HBV X protein, which may play a role in the Fas/FasL pathway-mediated apoptosis of HBV-infected liver cells (18, 19). Because SHP is expressed in large quantities in the liver (31), we examined a potential cross-talk between SHP and Nur77 and the effects of SHP in Nur77-mediated hepatic cell death in the present investigation. First, we tested whether SHP affects the transcriptional activity of Nur77. When HepG2 cells were treated with phorbol 12-myristate-13-acetate (PMA) and ionomycin, well-known stimulators of Nur77, the activity of reporter genes encoding Nur77 response element (NurRE) and NGFI-B response element (NBRE) were stimulated by approximately 30% and 300%, respectively. SHP inhibited the transcriptional activity of Nur77 induced by PMA/ionomycin in a dose-dependent manner. Transfection of equal or higher amounts of empty vectors did not decrease the reporter activities, suggesting that the decreases by SHP were not due to nonspecific transfection artifacts (Fig. 1AGo). Because the low inducibility of reporter gene activity by PMA/ionomycin in HepG2 cells may represent high-level expression of endogenous SHP, we introduced antisense (AS)-SHP into the cells. As expected, when AS-SHP was introduced, the basal activity and inducibility by PMA/ionomycin of the NurRE-Luc and NBRE-Luc reporters were increased (Fig. 1BGo). These results suggest that SHP may regulate the basal expression level as well as the transcriptional inducibility of Nur77 in HepG2 cells.



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Fig. 1. Transcriptional Activity of Nur77 Is Repressed by SHP in HepG2 Hepatoma Cells

A, Expression of SHP represses transcriptional activity of Nur77. NurRE-Luc (0.3 µg) or NBRE-Luc (0.3 µg) was cotransfected with the indicated amount of expression plasmid for SHP, pcDNA3-HA-SHP, or empty vector (EV) into HepG2 cells. B, Expression of AS-SHP enhances transcriptional activity of Nur77. NurRE-Luc (0.3 µg) or NBRE-Luc (0.3 µg) was cotransfected with the indicated amount of expression plasmid encoding AS-SHP, pcDNA3-AS-SHP. After being incubated overnight, the transfected cells were treated with or without PMA (10 ng/ml) and ionomycin [(Iono) 0.5 µM] for 6 h, after which cell lysates were obtained and analyzed. Luciferase activity was normalized for transfection efficiency by corresponding ß-gal activity. Data shown are the mean ± SD of three independent determinations. Cell lysates (50 µg) obtained from the transient transfection of 200 ng each empty vector (EV) or pcDNA3-AS-SHP were analyzed with anti-SHP antibody by Western blot analysis, and the result is shown as inset of upper panel.

 
To confirm the inhibition of transcriptional activity of Nur77 by SHP, we employed CV-1 cells, which do not express a significant amount of NRs. In CV-1 cells, the NurRE and NBRE reporter activities were induced only when Nur77 was exogenously expressed. The transcriptional activity of Nur77 was repressed by SHP, in a dose-dependent manner (Fig. 2AGo). Similarly, transcriptional activity of Nor-1 as well as Nurr1 in both NurRE-Luc and NBRE-Luc reporters were decreased by SHP (Fig. 2BGo). Because putative Nur77 binding sequences are present in the cytochrome P450 (CYP)17 promoter, we examined whether SHP inhibited transcriptional activity of Nur77 on the natural promoter (32). As expected, Nur77 increased the promoter activity, whereas cotransfection of SHP almost completely repressed the Nur77-induced CYP17 promoter (–1021)-Luc activity (Fig. 2CGo).



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Fig. 2. Transcriptional Activities of Nur77, Nor-1, and Nurr1 Are Repressed by SHP in CV-1

A, Transcriptional activity of Nur77 is repressed by SHP in CV-1 cells. NurRE-Luc (0.1 µg) or NBRE-Luc (0.1 µg) was cotransfected with 50 ng pECE-Nur77 and the indicated amount of pcDNA3-HA-SHP into CV-1 cells. B, Transcriptional activities of Nor-1 and Nurr1 are repressed by SHP. NurRE-Luc (0.1 µg) or NBRE-Luc (0.1 µg) was cotransfected with 50 ng pCMX-Nor-1 or pCMX-Nurr1 and the indicated amount of pcDNA3-HA-SHP into CV-1 cells. C, The CYP17 promoter activity is repressed by SHP. The CYP17 promoter-Luc (0.1 µg) was cotransfected with 50 ng pECE-Nur77 and the indicated amount of pcDNA3-HA-SHP into CV-1 cells. After 24 h of incubation, cell lysates were obtained and analyzed. Luciferase activity was normalized for transfection efficiency by corresponding ß-gal activity. Data shown are the mean ± SD of three independent determinations.

 
Transactivation Function of N-Terminal Domain of Nur77 Is Inhibited by SHP
To elucidate the mechanism by which SHP represses the transcriptional activity of Nur77, we first tested whether SHP interferes with the transactivation function of Nur77. We constructed several Gal4-DBD-fused Nur77 mutants and examined their transactivation function using the Gal4-tk-Luc containing Gal4 DNA binding sequences (Fig. 3AGo). The transactivation function of Nur77 was retained in N-terminal activation domain in both HepG2 and CV-1 cells, which was consistent with previous reports (6). The transactivation function of Nur77ND, which contained the N-terminal activation domain and DBD, was dramatically repressed by coexpression of SHP in both HepG2 and CV-1 cells (Fig. 3BGo). Recently, 6-MP was shown to enhance transactivation and coactivator recruitment of Nur77 family genes through the AF-1 domain (11). Therefore, we tested whether the repression of the transactivation of Nur77 by SHP was recovered by 6-MP treatment. As shown in Fig. 3CGo, 6-MP recovered the reporter activity that was repressed by SHP in a dose-dependent manner. 6-MP further increased the transcriptional activity of Nur77ND; however, SHP repressed the increased Nur77ND activity in a dose-dependent manner. These results suggested that SHP represses the transcriptional activity of Nur77 through the N-terminal transactivation domain.



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Fig. 3. Transactivation Function of N-Terminal Domain of Nur77 Is Inhibited by SHP

A, Schematic representation of Gal4-Nur77 constructs. TAD, Transactivation domain. B, Transactivation function of N-terminal domain of Nur77 is repressed by SHP. Gal4-tk-Luc (0.1 µg) was cotransfected with 10 ng each Gal4-Nur77Full, Gal4-Nur77NT, Gal4-Nur77ND, or Gal4-Nur77Hga into HepG2 (upper panel) or CV-1 (lower panel) cells. After transfected cells were incubated overnight, cell lysates were obtained and analyzed. C, 6-MP and SHP reciprocally modulate transcriptional activity of Gal4-Nur77ND. Gal4-tk-Luc (0.1 µg) was cotransfected with 10 ng Gal4-Nur77ND and 200 ng SHP expression vector (left panel) or the indicated amount of SHP expression vector (right panel) into HepG2 cells. Transfected cells were treated with the indicated concentrations of 6-MP for 24 h after which cell lysates were obtained and analyzed. Luciferase activity was normalized for transfection efficiency by corresponding ß-gal activity. Data shown are the mean ± SD of three independent determinations.

 
SHP Interacts and Colocalizes with Nur77 in Vivo
Next, we examined whether physical interaction of SHP with Nur77 was required to repress the transcriptional activity of Nur77 by SHP. When Nur77 was immunoprecipitated using specific anti-Nur77 antibodies, SHP was coprecipitated in HepG2 cells. Reciprocally, Nur77 was coimmunoprecipitated by anti-SHP antibodies, indicating that these proteins are physically associated in vivo (Fig. 4AGo). The result was further confirmed by coimmunoprecipitation of hemagglutinin (HA)- or FLAG-tagged proteins (Fig. 4BGo). An immunofluorescence study showed that both SHP and Nur77 were expressed mainly in the nucleus (Fig. 4CGo). When these proteins were coexpressed, their expression was colocalized in the nucleus as shown in Fig. 4CGo, further indicating that SHP and Nur77 interact in the nucleus. To show whether this interaction occurred on the Nur77-binding DNA sequences in the promoter of Nur77 target genes, we performed chromatin immunoprecipitation (ChIP) assay using the CYP17 promoter, which contains two putative Nur77-binding sequences (32). As shown in Fig. 4DGo, anti-Nur77 antibody precipitated the CYP17 promoter, demonstrating that Nur77 bound to this promoter. Anti-SHP antibodies also precipitated the CYP17 promoter, indicating that SHP is associated with this promoter probably by interacting with Nur77. Finally, we examined whether SHP localized in the cytoplasm when Nur77 translocated into the cytoplasm from the nucleus after PMA/ionomycin treatment. When HepG2 cells were treated with PMA/ionomycin, Nur77 was detected in both the nuclear and cytosolic fraction, indicating that some, but not all, Nur77 proteins was translocated into the cytoplasm (Fig. 4EGo). Interestingly, SHP appeared in the cytosolic fraction after PMA/ionomycin treatment, suggesting that SHP may translocate into cytoplasm due to physical association with Nur77.



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Fig. 4. Nur77 Interacts and Colocalizes with SHP in Vivo

A, Physical association of Nur77 with SHP in HepG2 cells. Whole-cell lysates (2 mg) were obtained from HepG2 cells and immunoprecipitated (IP) with normal IgG, anti-Nur77 (left panel), or anti-SHP (right) antibodies, and then analyzed with anti-SHP or anti-Nur77 antibodies by Western blot (W) analysis. Expression of Nur77, SHP, and {alpha}-tubulin was analyzed by Western blot nalysis as control. B, HEK293 cells were transfected with 2 µg p3XFLAG7.1-Nur77 and 2 µg pcDNA3-HA-SHP. After 24 h of incubation, cell lysates were obtained. Cell lysates (500 µg) were immunoprecipitated with anti-FLAG antibody and then analyzed with anti-HA antibody by Western blot analysis. Expression of FLAG-Nur77 and HA-SHP was analyzed by Western blot analysis as control. C, Nur77 and SHP colocalize in nucleus. HEK293 cells were transfected with 0.5 µg GFP-SHP alone or 0.5 µg each Red-Nur77 and GFP-SHP. After 24 h of transfection, cells were fixed and visualized by immunofluorescence microscopy. 4',6-diamino-2-phenylindole (DAPI) was used to stain nuclei. D, Nur77 and SHP interact on the promoter of CYP17 gene. Schematic presentation of the ChIP assay (upper panel). HepG2 cells were transfected with CYP17-promoter (–1021)-Luc. Nuclear lysates were incubated with normal IgG, anti-Nur77, or anti-SHP antibodies. Immunoprecipitated DNA was amplified by PCR using primers specific to the flanking regions of the Nur77-binding sites on the CYP17 promoter. C represents PCR amplification of nuclear lysates, which was done as positive control (lower panel). E, Intracellular distribution of Nur77 and SHP in HepG2 cells. HepG2 cells were treated with or without P/I [PMA (10 ng/ml) and ionomycin (0.5 µM)] for 24 h. At the end of incubation, whole-cell lysates and nuclear and cytosolic fractions were obtained and assayed for Nur77 and SHP expression by Western blot analysis. One representative of at least three independent experiments with similar results is shown. EV, Empty vector.

 
SHP Competes with Nur77 for Recruiting Coactivator cAMP-Response Element-Binding Protein (CREB)-Binding Protein (CBP)
The physical interaction of SHP with Nur77 may result in repression of the transcriptional function of Nur77 by interfering with efficient coactivator recruitment. Transcriptional coactivators, CBP and its homolog p300, SRC-1 and its family members, and activating signal cointegrator-2 (ASC-2), were shown to be essential for the activation of a large number of transcription factors, including many members of the NR superfamily (33, 34). The N-terminal region of Nur77 is implicated in the transactivation function by recruiting coactivators such as SRC and p300 (6). However, the binding of CBP with Nur77 or SHP has not been demonstrated experimentally. Given the possibility that coactivators are involved in the cross-talk between Nur77 and SHP, we examined whether SHP and Nur77 interacted with CBP. As shown in Fig. 5AGo, anti-CBP antibodies, but not normal IgG, immunoprecipitated both SHP and Nur77 in HepG2 cells, indicating that CBP is physically associated with these proteins in vivo. These interactions were further confirmed by immunoprecipitation of HA- or FLAG-tagged proteins (Fig. 5BGo). When the amount of SHP expression was increased, the binding of Nur77 with CBP was decreased (Fig. 5CGo). These results indicate that SHP and Nur77 bind to CBP competitively. Next, we tested whether exogenously introduced coactivator recovered the repression of Nur77 function caused by SHP. As expected, expression of coactivators such as CBP, p300, ASC-2, and PGC-1, recovered the transcriptional activity of Nur77 that was repressed by SHP (Fig. 6Go).



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Fig. 5. SHP and Nur77 Compete for Binding to Coactivator CBP

A, SHP and Nur77 interact with CBP in HepG2 cells. Whole-cell lysates (2 mg) were obtained from HepG2 cells and immunoprecipitated (IP) with normal IgG or anti-CBP antibodies and then analyzed with anti-SHP or anti-Nur77 antibodies by Western blot (W) analysis. Expression of SHP, Nur77, and CBP was analyzed by Western blot analysis as control. B, SHP and Nur77 interact with CBP in HeLa cells. HeLa cells were transfected with 2 µg pcDNA3-HA-SHP (left panel) or 2 µg p3XFLAG7.1-Nur77 (right panel) with 2 µg pRC-RSV-CBP. After 24 h of incubation, cell lysates were obtained. Cell lysates (500 µg) were immunoprecipitated with anti-HA or anti-FLAG antibody and then analyzed with anti-CBP antibody by Western blot analysis. Expression of HA-SHP, FLAG-Nur77, CBP, and {alpha}-tubulin was analyzed as control. C, Expression of SHP reduces association between Nur77 and CBP. HeLa cells were transfected with DNA mixture (3 µg/well) containing 0.6 µg pEBG-Nur77 and 0.6, 1.2, 1.8, or 2.4 µg pcDNA3-HA-SHP together with pBluescript. After 24 h of incubation, cell lysates were obtained. Cell lysates (500 µg) were immunoprecipitated with anti-glutathione-S-transferase (GST) antibody, and then analyzed with anti-CBP antibody by Western blot analysis. Expression of HA-SHP and GST-Nur77 was analyzed by Western blot analysis as control. One representative of at least three independent experiments with similar results is shown.

 


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Fig. 6. Expression of Coactivators Recovers Transcriptional Activity of Nur77 That Is Repressed by SHP

NurRE-Luc (0.1 µg) was cotransfected with 50 ng pECE-Nur77 and/or 50 ng pCDNA3-HA-SHP, in the presence or absence of the indicated amount of expression vectors for coactivators, pRC-RSV-CBP, pcDNA3-p300, or pcDNA3-HA-ASC-2 into CV-1 cells. After transfected cells were incubated overnight, cell lysates were obtained and analyzed. Luciferase activity was normalized for transfection efficiency by corresponding ß-gal activity. Data shown are the mean ± SD of three independent determinations.

 
Previous study demonstrated that a loop region containing extra amino acids between helices H6 and H7 in the LBD of SHP plays an important role in repression of NRs (35). To examine whether this loop region of SHP was involved in the repression of Nur77, a SHP mutant lacking the loop, SHP{Delta}Loop, was tested for the repressive function (Fig. 7AGo). The wild-type SHP abolished most of the transcriptional activity of Nur77; however, the SHP{Delta}Loop did not repress the Nur77 function at all (Fig. 7BGo). The SHP{Delta}Loop bound to Nur77 as efficiently as the wild-type SHP (Fig. 7CGo), indicating that the loop region is important in the repression of Nur77 although it is not required for the interaction with Nur77.



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Fig. 7. SHP Mutant that Lacks the Loop Region between Helices H6 and H7 Does Not Repress Transcriptional Activity of Nur77

A, Schematic representation of SHP and its mutant that lacks the loop region between helices H6 and H7, SHP{Delta}Loop. B, The SHP{Delta}Loop does not repress transcriptional activity of Nur77. NurRE-Luc reporter (0.1 µg) was cotransfected with 50 ng pECE-Nur77, 50 ng pcDNA3-HA-SHP, or the indicated amount of pcDNA3-HA-SHP{Delta}Loop into CV-1 cells. After transfected cells were incubated overnight, cell lysates were obtained and analyzed. Luciferase activity was normalized for transfection efficiency by corresponding ß-gal activity. Data shown are the mean ± SD of three independent determinations. C, The SHP{Delta}Loop interacts with Nur77. HEK293 cells were transfected with 2 µg each pcDNA3-HA-SHP or pcDNA3-HA-SHP{Delta}Loop together with 2 µg FLAG-Nur77. After 24 h of transfection, cell lysates were obtained. Cell lysates (500 µg) were immunoprecipitated (IP) with anti-FLAG antibody and then analyzed with anti-HA antibody by Western blot (W) analysis. Expression of FLAG-Nur77, HA-SHP{Delta}Loop, and HA-SHP was analyzed by Western blot analysis as control. One representative of at least three independent experiments with similar results is shown.

 
SHP Inhibits Anti-Fas-Mediated Apoptosis of Hepatic Cells
The Fas/FasL signaling pathway is important in mediating inflammatory cell death of hepatocytes (36, 37, 38). Shin et al. (39) showed that triggering of Fas using anti-Fas antibody, CH11, induced apoptotic cell death in liver cells. To elucidate the role of cross-talk between Nur77 and SHP, we employed SNU354 and HepG2 cell lines, which have a different susceptibility for CH11-induced apoptosis (39). When SNU354 cells were treated with CH11, a large portion of cells underwent apoptosis (Fig. 8AGo). During the course of apoptosis of SNU354, expression of Nur77 mRNA was largely increased. Similarly, expression of Bax, a proapoptotic gene, and NDG2, recently described as a downstream target gene of Nur77 in thymocytes, was induced after CH11 treatment (Fig. 8BGo). Consistent with the increase in transcripts, transcriptional activity of Nur77 was induced after CH11 treatment when measured by reporter gene analysis (Fig. 8CGo). The Nur77 protein that was induced by CH11 treatment was localized mainly in the nucleus, which was in contrast to the PMA/ionomycin treatment that induced partial translocation of Nur77 into the cytoplasm (Fig. 8DGo). These results indicate that the CH11-induced apoptosis of SNU354 is accompanied by the induction of expression as well as transactivation function of Nur77.



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Fig. 8. Nur77 Plays an Important Role in Anti-Fas Antibody-Induced Apoptosis of SNU354

A, Induction of apoptosis in SNU354 by IFN{gamma}/CH11-treatment. SNU354 cells were treated with vehicle (control) or IFN{gamma}/CH11 and stained with annexin V as described in Materials and Methods. Numbers indicate percentage of cells positively stained with annexin V. B, Expression of Nur77, Bax, and NDG2 during the course of IFN{gamma}/CH11-treatment. At the end of treatment with IFN{gamma}/CH-11, total RNA was prepared and analyzed for expression of the transcripts by RT-PCR. C, Transcriptional activity of Nur77 is induced by IFN{gamma}/CH11. NurRE-Luc (0.3 µg) was transfected into SNU354, and transfected cells were treated with IFN{gamma}/CH11 as described in Materials and Methods. Luciferase activity was normalized for transfection efficiency by corresponding ß-gal activity. Data shown are the mean ± SD of three independent determinations. D, Expression of Nur77 protein is induced by IFN{gamma}/CH11 treatment, and it localizes in the nucleus. SNU354 cells were treated with vehicle or IFN{gamma}/CH11. P/I [PMA (100 ng/ml) and ionomycin (0.5 µM)] was treated for 24 h for comparison. At the end of incubation, whole-cell lysates and nuclear and cytoplasmic fractions were obtained and assayed for Nur77 expression by Western blot analysis. E, Repressive function of the DN-Nur77 in transcriptional activity of Nur77. NBRE-Luc (0.1 µg) was cotransfected with 10 ng pECE-Nur77 and/or 10 ng pcDNA3-DN-Nur77 into CV-1 cells. After overnight incubation, cell lysates were obtained and analyzed. Luciferase activity was normalized for transfection efficiency by corresponding ß-gal activity. Data shown are the mean ± SD of three independent determinations. F, DN-Nur77 inhibits IFN{gamma}/CH11-induced apoptosis of SNU354. Two micrograms of the vector encoding DN-Nur77 or pcDNA3-DN-Nur77 or 2 µg empty vector (EV) were transfected into SNU354 cells. Transfected cells were treated with vehicle (control) or IFN{gamma}/CH11 and then stained with annexin V. G, DN-Nur77 inhibits expression of Bax transcripts induced by IFN{gamma}/CH11 treatment. SNU354 cells were transfected and treated with IFN{gamma}/CH11 as described in panel F. Total RNA was obtained and analyzed for the expression of Bax transcripts by RT-PCR. The expression of ß-actin was monitored as control. A representative result obtained from as least three independent experiments is shown.

 
To examine whether Nur77 plays an active role in the CH11-induced apoptosis, we transfected a eukaryotic expression vector encoding a dominant-negative (DN) Nur77 mutant into SNU354 cells. The repressive function of the DN-Nur77 was confirmed by reporter gene analysis (Fig. 8EGo). When the DN-Nur77 was transfected, the percentage of apoptotic cells, as well as the expression of Bax and NDG2, was dramatically reduced in comparison with the transfection of empty vector (Fig. 8Go, F and G). The result indicates that the induction of Nur77 plays a critical role in the IFN{gamma}/CH11-induced apoptosis of the SNU354 cells. In contrast, HepG2 cells were relatively insensitive to CH11-induced apoptosis in that only 15% of cells underwent apoptosis (Fig. 9AGo). The induction level of Nur77 was consistently less than that of SNU354 (Fig. 9BGo). Interestingly enough, expression of SHP was not detectable in SNU354, whereas a significant amount of SHP was expressed in HepG2 cells. Taken together, these results may indicate that the expression level of SHP is related to the transcriptional induction as well as apoptotic function of Nur77 in hepatoma cells.



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Fig. 9. Expression of SHP in Hepatoma Cell Lines that Have Differential Sensitivity to IFN{gamma}/CH11-Induced Apoptosis

A, HepG2 cells were not sensitive to IFN{gamma}/CH11-treatment. HepG2 cells were treated with vehicle (control) or IFN{gamma}/CH11 and stained with annexin V as described in Materials and Methods. Numbers indicate percentage of cells positively stained with annexin V. B, Expression of Nur77 and SHP in IFN{gamma}/CH11-treated SNU354 and HepG2. At the end of treatment with IFN{gamma}/CH11, total RNA was prepared and analyzed for expression of the transcripts by RT-PCR as described in Materials and Methods. One representative of at least three independent experiments with similar results is shown.

 
Finally, to confirm the role of SHP in the Nur77-mediated apoptosis, we established the SNU354 cell lines that expressed SHP. The SHP-expressing SNU354 stable cell line became resistant to IFN{gamma}/CH11 treatment when it was compared with the cell line that expressed empty vector (Fig. 10Go, A and B). Similar results were obtained from at least three different clones (data not shown). These results strongly suggest that SHP is an important regulator of Nur77-mediated apoptotic cell death of liver cells.



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Fig. 10. Expression of SHP Represses IFN{gamma}/CH11-Induced Apoptosis of SNU354

A, Expression of SHP in the SNU354 stable cell line. Whole-cell lysate was prepared and analyzed for expression of HA-SHP protein in the SNU354 stable cell lines expressing pcDNA3 (EV) and pcDNA3-HA-SHP using HA antibodies by Western blot analysis. B, SNU354 cell lines that stably express empty vector or SHP were treated with vehicle (control) or IFN{gamma}/CH11 and then stained with annexin V as described in Materials and Methods. Numbers indicate percentage of cells positively stained with annexin V. One representative of at least three independent experiments with similar results is shown.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SHP has been reported to function as a repressor of various NRs, including retinoic acid receptor/retinoic X receptor, estrogen receptor, PXR, and liver X receptor (23, 24, 25, 26, 27, 28, 29, 30). The cross-talk between SHP and NRs, which are abundantly expressed in the liver, may regulate the important metabolic processes occurring in the tissue, such as energy expenditure and the detoxification of various hazardous materials. In the present investigation, we provide evidence, for the first time, that cross-talk between SHP and Nur77 exists in the course of hepatic cell death, providing a potential link between lipid metabolism and inflammatory liver diseases that are mediated by two different subfamilies of NRs.

SHP interacts and represses transcriptional activity of various NRs through several different mechanisms. Here, we demonstrate that SHP binds coactivator CBP, thereby squelching the coactivator from Nur77, resulting in repression of the transcriptional function of Nur77. The mode of repression may be similar to that of SHP-induced transcriptional repression of GR and PXR (29, 30). SHP antagonizes transcriptional activity of GR and PXR by competing for coactivators PGC-1 and SRC-1, respectively (29, 30). The repression of Nur77 function induced by SHP was recovered by cotransfection of CBP/p300 and ASC-2 as well as PGC-1 (Fig. 6Go), suggesting that both receptors may bind competitively to a wide spectrum of coactivators, which may provide an intensive cross-talk between these two receptors. The facts that N-terminal region of Nur77 contains AF-1 activity recruiting coactivators (6) and that 6-MP, which activates transcriptional function of Nur77 through the N-terminal activation domain (11), recovers the repression induced by SHP (Fig. 3CGo), indicate that the N terminus of Nur77 is a target of the SHP-induced repression. For the cross-talk of these two receptors, physical association is required. This interaction may occur on the DNA binding sequences of the promoter of Nur77 target genes as shown by ChIP assays (Fig. 4DGo), further supporting the role of SHP in the repression of transcriptional activity of Nur77.

Thus far, the mechanism by which Nur77 induces apoptosis has not been clearly elucidated. Recently, it has been reported that, in response to apoptotic stimuli, Nur77 translocates from the nucleus to the cytoplasm where it targets mitochondria to lead cytochrome c release in some cells, such as prostate and lung cancer cells (7, 20). In these cells, the interaction of Nur77 with the Bcl-2 apoptotic machinery converts Bcl-2 from a protector to a killer (21). In this case, the apoptotic effect of Nur77 does not require its transcriptional activity or DNA binding. In contrast, Nur77 is localized mainly in the nucleus, and Bcl-2 does not antagonize Nur77-mediated apoptosis in the T cell hybridomas (22). Thus, the mitochondrial pathway is not the major apoptotic pathway in these cells. Indeed, a microarray analysis revealed that many genes, including FasL, TRAIL, NDG1, and NDG2, which activate known and novel apoptotic pathways, are induced in thymocytes carrying the Nur77 transgene (22). We observed that the transcriptional activity of Nur77 was largely enhanced during CH11 treatment in SNU354 cells, and it is exclusively localized in the nucleus (Fig. 8Go). Further, the DN-Nur77, which represses transactivation function of Nur77, diminished the CH11-induced apoptosis of SNU354 (Fig. 8Go). Interestingly, the expression of NDG2 was also increased in the CH11-induced apoptosis of SNU354 (Fig. 8BGo). Together, these results indicate that the transactivation function of Nur77 is required and plays a critical role in the regulation of the Fas/FasL-induced apoptotic pathway in the liver cells.

Acute liver failure due to hepatic apoptosis is a symptom of many different kinds of liver disease, such as infection with viral hepatitis, alcoholic hepatitis, and autoimmune hepatitis. Although the molecular pathways leading to the death of hepatocytes under various conditions are not well understood, recent studies suggest that death ligands of the TNF family such as FasL and TRAIL may play a crucial role (36, 37, 38). Previously, we reported that HBV X protein induces expression of FasL through activation of Nur77, which may contribute to the Fas/FasL pathway-induced liver injury (18, 19). In this report, we demonstrate that the Fas/FasL apoptotic pathway, triggered by antagonistic anti-Fas antibody, induces expression of Nur77, which plays a critical role in the apoptotic pathway (Fig. 8Go). In fact, FasL and TRAIL are also identified by others (22) as downstream target genes of Nur77 in thymocytes. From these observations, we speculate that SHP has a role in the protection of hepatocytes from Nur77-induced apoptosis that frequently occur in hepatitis and are induced by variety of endogenously or exogenously generated stimuli. Further, our results may provide an insight into the control of inflammatory liver diseases through regulation of hepatic apoptosis via cross-talk between NRs.

Recently, a series of mutations affecting the human SHP protein were identified in Japanese subjects with mild obesity (40). The mutant SHP proteins that have an abnormal C-terminal region were demonstrated to have less function than wild-type SHP in variety of biological functions such as inhibiting transcriptional function of estrogen-related receptor-{alpha} and BETA2 (41, 42). We demonstrate that the mutant SHPeT23, which is deleted from the middle receptor interaction domain to the entire C terminus, is less effective in inhibiting transcriptional activity of Nur77 and that it does not bind to CBP (data not shown). Mutations in SHP have been studied for a physiological basis of common metabolic conditions such as obesity and diabetes. Recently, it has been increasingly recognized that there are potential links between obesity, insulin resistance, and the liver diseases. For example, steatosis, accumulation of lipid droplet in liver, accelerates the progression of liver damage of chronic hepatitis C patients and is correlated with visceral obesity (43). Diabetes seems to occur frequently in patients infected by chronic hepatitis C virus (44). It is tempting, therefore, to study whether such mutations in SHP affect initiation and progression of inflammatory liver diseases such as alcoholic hepatitis and hepatic viral infections.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells, Reagents, and Antibodies
Human hepatocellular carcinoma cell line, HepG2 (ATCC HB 8065) and human cervical carcinoma cell line, HeLa (ATCC CCL-2), and green monkey kidney epithelial cell line, CV-1 (ATCC CCL-70) were obtained from the American Type Culture Collection. The human hepatocellular carcinoma cell line SNU354 was obtained from the Korean cell line bank. Hepatoma cells were maintained in either RPMI or MEM containing 10% fetal bovine serum at 37 C in an atmosphere of humidified incubator with 5% CO2 and 95% air. Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma Chemical Co. (St. Louis, MO). Ionomycine was obtained from Calbiochem (San Diego, CA). The other chemicals used were of the purest grade available from Sigma. Rabbit polyclonal anti-Nur77, goat polyclonal anti-SHP, anti-CBP, and anti-hemagglutinin (HA) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antagonistic anti-Fas antibody (CH11) and mouse anti-Nur77 antibody were obtained from MBL Co. (Nagoya, Japan) and PharMingen (San Diego, CA), respectively. Anti-FLAG antibodies were purchased from Sigma. Recombinant interferon-{gamma} (IFN{gamma}) was obtained from R&D Systems, Inc. (Minneapolis, MN)

Plasmids
The reporter genes, i.e. NurRE-pomc-Luc, NBRE-tk-Luc, Gal4-tk-Luc, and the CYP17-promoter (–1021)-Luc reporter constructs have been described elsewhere (18, 45, 46). Eukaryotic expression vectors for Nur77-related genes, i.e. pECE-Nur77, pCMX-Nor-1, pCMX-Nurr1, pcDNA3-DN-Nur77, pcDNA-AS-Nur77, green fluorescent protein (GFP)-SHP, and pEBG-Nur77 were described previously (18, 47). The FLAG-tagged full-length Nur77 was constructed by inserting PCR-amplified full-length Nur77 fragments into the EcoRI/BamHI site of p3XFLAG-7.1. The Gal4 DBDs fused with full-length or deleted Nur77 constructs were generated by inserting PCR-amplified Nur77 fragments into the EcoRI/BamHI site of PM (CLONTECH, Palo Alto, CA). The far-red fluorescent protein tagged-Nur77, Red-Nur77, was constructed by inserting corresponding PCR-amplified fragments into the EcoRI/BamHI sites of pHcRed1-C1 (CLONTECH). All the construction was confirmed by sequencing. Eukaryotic expression vectors for SHP-related genes, i.e. HA-tagged mouse SHP expression vectors, pcDNA3-HA-SHP, and pcDNA-HA-SHP{Delta}Loop, were described previously (36). Eukaryotic expression vectors for coactivators, i.e. CBP, p300, PGC-1, and ASC-2, were described previously (35, 48, 49, 50).

Transient Transfection and Reporter Gene Assay
HeLa cells (5 x 105 cells per well) were seeded in six-well plates and incubated overnight. The cells were transfected with 2 or 3 µg expression vectors using Polyfect (QIAGEN, Chatsworth, CA) according to the manufacturer’s instructions. After 24 h of transfection, cells were lysed and assayed for Western blot analysis as described below. For reporter gene assays, HepG2 cells (1.5 x 105 cells per well) were seeded in a 12-well culture plate and transfected with 0.3 µg reporter plasmid, 0.25 µg ß-galactosidase (ß-gal) expression vector, in the presence or absence of receptor expression vectors using Lipofectamine-Plus (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. For transient transfection of CV-1 cells, a modified calcium phosphate precipitation procedure was used. Cells (5 x 104 cells per well) were seeded in a 24-well culture plate and transfected with DNA mixture (1 µg/well) containing 0.1 µg reporter plasmid, 0.2 µg ß-gal expression vector, and various combinations of receptor expression vectors with carrier DNA (pBluescript) (18, 30). After 24 h of transfection, cells were lysed in the cell culture lysis buffer, and luciferase activity was determined using an Analytical luminescence luminometer. Luciferase activity was normalized for transfection efficiency by the corresponding ß-gal activity. For statistical analysis, one-way ANOVA was performed using GraphPad Instat (GraphPad Software, San Diego, CA). A value of P < 0.05 was considered statistically significant.

Western Blot Analysis/Immunoprecipitation
Cells were lysed in a lysis buffer containing 50 mM Tris (pH 8.0), 100 mM NaCl, 5 mM EDTA, 1% Nonidet P-40 (NP-40), and protease inhibitors (Roche Diagnostics, Indianapolis, IN). After a 30-min incubation on ice, cell lysates were centrifuged at 14,000 x g for 10 min at 4 C, and the clear supernatants were saved as whole-cell lysate. For subcellular fractionation, cells were resuspended in a buffer containing 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, and protease inhibitor, for 30 min on ice. After addition of the 10% NP-40, cell lysate was sheared by 10 passages though a 22-gauge needle and centrifuged at 4000 x g for 15 min at 4 C. The resulting supernatant was saved as the cytosolic fraction. The pellet was resuspended in a buffer containing 20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, and protease inhibitors, by rocking at 4 C for 30 min. After centrifugation at 14,000 x g for 10 min, resulting supernatant was saved as the nuclear fraction. Western blot analysis using specific anti-HA, anti-CBP, and anti-FLAG antibodies was performed as described previously (45). For immunoprecipitation, 500 µg of whole-cell lysate were incubated overnight with 1 µg antibodies with agitation at 4 C. The resulting immune complex was precipitated by adding 20 µl protein-G agarose slurry, washed four times with the lysis buffer, subjected to 8% SDS-PAGE, and transferred to nitrocellulose membrane. The membrane was probed with the anti-CBP, anti-HA, and anti-FLAG antibodies. Protein concentrations of the lysates were quantified by bicinchoninic acid assay (Pierce Chemical Co., Madison, WI).

Immunofluorescence
Human embryonic kidney (HEK)293 cells (1 x 105 cells per well) were plated in 12-well plates and incubated overnight. Cells were transfected with 0.5 µg of GFP-SHP alone or 0.5 µg each Red-Nur77 and GFP-SHP using Polyfect. After 24 h of transfection, cells were fixed with 50% acetone-50% methanol for 5 min and then visualized by immunofluorescence microscopy (Olympus Corp., Lake Success, NY). 4',6-Diamidino-2 phenylindole was used to stain nuclei.

ChIP Assays
HepG2 cells (5 x 106 cells per dish) were seeded in 100-mm dishes and incubated overnight. The cells were transfected with 8 µg plasmid encoding CYP17-promoter using LipofectaminePlus. After 24 h of transfection, cells were cross-linked in 1% formaldehyde and resuspended in a buffer containing 1% NP-40, 25 mM Tris-HCl, pH 7.4, 5 mM MgCl2, and protease inhibitors. The nuclei were precipitated and lysed in lysis buffer containing 1% sodium dodecyl sulfate (SDS), 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, and protease inhibitors. Nuclear lysates were sonicated, and lysates were immunoprecipitated using specific anti-Nur77 and anti-SHP of normal IgG antibodies for 24 h at 4 C. The immunoprecipitates were successively washed with low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, and Tris-EDTA buffer. Immune complexes were eluted with elution buffer containing 1% SDS and 0.1 M NaHCO3, and formaldehyde cross-linking was reversed by adding 5 M NaCl. DNA was extracted and amplified by PCR using specific primers corresponding to the flanking regions of the Nur77 binding sites on the CYP17-promoter (forward, 5'-TGAAAGCCTATGACTTCT-3'; and reverse, 5'-CTGTCAAAGAGACTTCAG-3'). PCR products were resolved in 1.2% agarose gel.

RT-PCR
Total RNA was prepared using the QIAGEN RNeasy kit, and single-stranded DNA was synthesized from RNA in a reaction mixture containing 100 ng of random hexamer and 200 U of murine Moloney leukemia virus reverse transcriptase (Invitrogen). PCR was performed as previously described with specific primers for Nur77 (forward, 5'-CGACCCCCTGACCCCTGAGTT-3'; reverse, 5'-GCCCTCAAGGTGTTGGAGAAGT-3'), SHP (forward, 5'-GCCTTCCTCAGGAACCTGCC-3'; reverse, 5'-GGAGGCCTGGCACATC-3'), AS-SHP (forward, 5'-CCAGAAGGACTCCAGACAGC-3'; reverse, 5'-CTATGTGCACCT CATCGCAC-3'), Bax (forward, 5'-CAGCTCTGAGCAGATCATG AAGACA-3'; reverse, 5'-GCCCATCTTCTTCCAGATGGTGAGC-3'); NDG2 (forward, 5'-CCTTCAGTTCATCACCA ACA-3'; reverse, 5'-AAG AAAAACTCGCAAACACC-3'), and ß-actin (forward, 5'-CGTGGGCCGCCC TAGGCACCA-3'; reverse, 5'-TTGGCCTTAGGGTTCAGGGGGG-3') (18, 45). The genes were analyzed under conditions in which PCR products were exponentially amplified.

Analysis of Apoptosis Using Annexin V
To establish stable HCC cell line expressing AS-SHP or SHP, pcDNA3-AS-SHP and pcDNA3-HA-SHP were transfected into HepG2 and SNU354, respectively, using LipofectaminePlus. Positive clones were selected using G418 (800 and 200 µg/ml for HepG2 and SNU354, respectively) (Invitrogen). Wild-type or stably transfected HepG2 and SNU354 cells were seeded at 2 x 105 cells per dish in 60-cm2 dishes. After overnight incubation, cells were treated with IFN{gamma} (250 U/ml) for 36 h and then subsequently treated with CH11 (0.25 µg/ml) for another 36 h as described previously (39). At the end of incubation, cells were carefully collected and stained with propidium iodide and annexin V-fluorescein isothiocyanate conjugate (CLONTECH). Fluorescent intensity was measured with a FACStar PLUS flow cytometer (Becton Dickinson, Mountain View, CA).


    ACKNOWLEDGMENTS
 
We thank Dr. Thomas Perlmann for providing us Nurr1 and Nor-1 expression vectors. We also thank Drs. Anastasia Kralli and Jaewoon Lee for PGC-1 and ASC-2 expression vectors, respectively. The CYP17-promoter-Luc was kindly provided by Dr. Anita Payne.


    FOOTNOTES
 
This work was supported by grants from the Ministry of Science and Technology (M1-0312-04-0002 and M1-0311- 00-0089) and a grant of the 2003 Good Health R&D Project (03-PJ1-PG3-20900-0038) from the Ministry of Health and Welfare.

First Published Online December 29, 2004

Abbreviations: AF, Activation function; AS, antisense; ASC, activating signal cointegrator; ChIP, chromatin immunoprecipitation; CBP, CREB-binding protein; CYP, cytochrome P450; DBD, DNA-binding domain; DN, dominant negative; FasL, Fas ligand; ß-gal, ß-galactosidase; GFP, green fluorescent protein; GR, glucocorticoid receptor; HA, hemagglutinin; HBV, hepatitis B virus; IFN, interferon; LBD, ligand-binding domain; 6-MP, 6-mercaptopurin; NBRE, NGFI-B response element; NDG, Nur77 downstream gene; NP-40, Nonidet P-40; NR, nuclear receptor; NurRE, Nur77 response element; PGC, peroxisomal proliferator-activated receptor-{gamma} coactivator; PXR, pregnane X receptor; SHP, small heterodimer partner; SRC, steroid receptor coactivator; TRAIL, TNF-related apoptosis inducing ligand.

Received for publication May 20, 2004. Accepted for publication December 20, 2004.


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