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Cancer Research 67, 3616, April 15, 2007. doi: 10.1158/0008-5472.CAN-06-4020
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

SAG/ROC2/Rbx2 Is a Novel Activator Protein-1 Target that Promotes c-Jun Degradation and Inhibits 12-O-Tetradecanoylphorbol-13-Acetate–Induced Neoplastic Transformation

Qingyang Gu, Mingjia Tan and Yi Sun

Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan

Requests for reprints: Yi Sun, Department of Radiation Oncology, University of Michigan, 4304 CCGC, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0936. Phone: 734-615-1989; Fax: 734-647-9654; E-mail: sunyi{at}umich.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
SAG (sensitive to apoptosis gene) was first identified as a stress-responsive protein that, when overexpressed, inhibited apoptosis both in vitro and in vivo. SAG was later found to be the second family member of ROC1 or Rbx1, a RING component of SCF and DCX E3 ubiquitin ligases. We report here that SAG/ROC2/Rbx2 is a novel transcriptional target of activator protein-1 (AP-1). AP-1 bound both in vitro and in vivo to two consensus binding sites in a 1.3-kb region of the mouse SAG promoter. The SAG promoter activity, as measured by luciferase reporter assay, was dependent on these sites. Consistently, endogenous SAG is induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) with an induction time course following the c-Jun induction in both mouse epidermal JB6-Cl.41 and human 293 cells. TPA-mediated SAG induction was significantly reduced in JB6-Cl.41 cells overexpressing a dominant-negative c-Jun, indicating a requirement of c-Jun/AP-1. On the other hand, SAG seemed to modulate the c-Jun levels. When overexpressed, SAG remarkably reduced both basal and TPA-induced c-Jun levels, whereas SAG small interfering RNA (siRNA) silencing increased substantially the levels of both basal and TPA-induced c-Jun. Consistently, SAG siRNA silencing reduced c-Jun polyubiquitination and blocked c-Jun degradation induced by Fbw7, an F-box protein of SCF E3 ubiquitin ligase. Finally, SAG overexpression inhibited, whereas SAG siRNA silencing enhanced, respectively, the TPA-induced neoplastic transformation in JB6-Cl.41 preneoplastic model. Thus, AP-1/SAG establishes an autofeedback loop, in which on induction by AP-1, SAG promotes c-Jun ubiquitination and degradation, thus inhibiting tumor-promoting activity of AP-1. [Cancer Res 2007;67(8):3616–10]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Activator protein-1 (AP-1) transcription factor is a dimer consisting of 18 different combinations of Jun-Jun or Jun-Fos proteins. The Jun family of proteins includes c-Jun, JunB, and JunD, whereas the Fos family of proteins includes c-Fos, FosB, Fra-1, and Fra-2 (1). The c-jun and c-fos genes are inducible by a broad range of extracellular stimuli. On activation, AP-1 binds to 12-O-tetradecanoylphorbol-13-acetate (TPA) response elements or AP-1 binding sites, 5'-TGAG/CTCA-3', to transactivate many effector genes, thus regulating cell proliferation, tumor promotion, cell cycle, growth arrest, and apoptosis (1–3). The analysis of cells and mice deficient in individual AP-1 proteins has identified several physiologically relevant AP-1 target genes, including cyclin D1, p16, p19, p53, p21, and Fas ligand (1). Activation of cyclin D1 and inhibition of p53 and p21 would promote cell cycle progression from G1 to S to induce cell proliferation.

AP-1 was first considered as a mediator of tumor promotion by its ability to alter gene expression in response to tumor promoters, such as TPA and UV irradiation (3). Indeed, TPA, UV, as well as reactive oxygen species activate AP-1 (4–6). In mouse epidermal JB6 cell variants, representing from early to late stages of tumor promotion, the AP-1 activity is progressively elevated (7). On the other hand, cell growth and neoplastic transformation was significantly impaired on blockage of AP-1 activity by either pharmacologic inhibitors, such as glucocorticoid, retinoic acid, or molecular biological inhibitors, including dominant-negative c-Jun (TAM67), dominant-negative phosphatidylinositol-3 kinase (p85), and dominant-negative extracellular signal-regulated kinase 2 (Erk2; refs. 8–13). Moreover, acquisition of a tumor promotion-resistant phenotype is associated with a loss of responsiveness to tumor promoter-induced AP-1 activation (14), whereas rescuing such a response by introducing wild-type (WT) Erk2 converts tumor promotion-resistant phenotype to tumor promotion-sensitive phenotype (15).

SAG (sensitive to apoptosis gene) was initially cloned by differential display as a redox-inducible gene that encodes an evolutionarily conserved RING finger protein (16, 17). Further characterization of SAG revealed that SAG is a dual-function protein with antioxidant activity when acting alone or E3 ubiquitin ligase activity when complexed with other ligase components (17, 18). Recently, ROC1/Rbx1/Hrt1, a family member of SAG and a component of SCF (Skp1, cullin1, and F-box protein) and DCX (DDB1, cullin 4A, and X-box protein) E3 ubiquitin ligases was implicated in ubiquitination and degradation of c-Jun (19–23). However, it has not been shown previously that SAG, through binding to Cul-1 and Cul-4A (20), will replace ROC1/Rbx1 to form a SAG-SCF E3 ligase and promote c-Jun degradation. We report here that SAG is induced by TPA via AP-1–mediated transactivation. On the other hand, AP-1 component, c-Jun, is a SAG substrate. When overexpressed, SAG remarkably reduced both basal and TPA-induced c-Jun levels, whereas SAG small interfering RNA (siRNA) silencing significantly increased the levels of both endogenous and TPA-induced c-Jun. SAG silencing inhibited c-Jun polyubiquitination and blocked Fbw7-induced c-Jun degradation. Finally, SAG overexpression within physiologic levels inhibited, whereas SAG silencing enhanced, the TPA-induced neoplastic transformation in JB6-Cl.41 cells. Thus, it seems that c-Jun-SAG constitutes a negative feedback loop as a cellular defensive mechanism to minimize the prolonged effect of AP-1 activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Human HeLa, 293, and H1299 cells were cultured in DMEM containing 10% fetal bovine serum (FBS). Mouse JB6-Cl.41 cells were cultured in DMEM containing 5% FBS. JB6-Cl.41 cells expressing TAM67 (24) were cultured in 5% DMEM containing G418 (400 µg/mL).

Promoter cloning, luciferase reporter construction, and luciferase activity assay. A 1.3-kb fragment upstream of mouse SAG translational initiation site was cloned by PCR with primers mSAG-P01 (5'-GGGGTACCAGCCTTGGTTGCCCGAAAC-3') and mSAG-P02 (5'-CCGCTCGAGGGCGGCGGCGCAGAACGG-3'). The fragment was then gel purified and subcloned into pGL3-basic luciferase reporter (Promega, Madison, WI) at the KpnI/XhoI or KpnI/HindIII site with subsequent sequencing confirmation as described (25). The luciferase reporter was designated as mSAG-P1.3 or 7AP (for containing seven putative AP-1 binding sites). This construct was used as the template to generate a series of deletion or site-directed mutants, including 5AP (containing five AP-1 sites) with the primer set of AP1-3-KpnI (5'-GGTACCGAGGGATGACTCAGTGGT-3') and mSAG-P02; 4AP (containing four AP-1 sites) with primer set of D3 (5'-GGTACCGTGGTTAAAACCATGGTCTG-3') and mSAG-P02; 2AP (containing two AP-1 sites) with the primer set of AP1-6-KpnI (5'-GGTACCCCTCGTGACGTCACTGGC-3') and mSAG-P02; 1AP (containing one AP-1 site) with the primer set of AP1-7-KpnI (5'-GGTACCGCGCCATCCAATCATCGC-3') and mSAG-P02; and 0AP (with all AP-1 sites deleted) with the primer set of D5 (5'-GGTACCTCGCCGTCTGGCTCCGCCCG-3') and mSAG-P02. A T->A substitution at AP1-3 and AP1-6 primers was introduced to generate Mu-5AP and Mu-2AP constructs with AP-1 binding site abolished. To generate the 1.3-kb promoter construct with AP-1 sites 3 and 6 mutated (Mu-3,6), the QuickChange Multi Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) was used, following the manufacturer's instruction. The sequence of the primers used is Sag-AP1-mu3-01 (5'-TGTAGTGTCTGGAGGGAAGACTCAGTGGTTAAAAC-3') and Sag-AP1-mu6-01 (5'-CCCAGCCCCTCGAGACGTCACTGGC-3'). To measure the promoter activity of the mSAG-P1.3 and its deletion or site-directed mutants, HeLa cells were seeded in a 96-well plate at a density of 18,000 cells per well. Cells were transiently cotransfected, using TR01 transfection reagent (America Pharma Source, Gaithersburg, MD), with these luciferase reporters individually, along with a plasmid expressing Renilla luciferase (Promega) as a control for transfection efficiency. Forty hours post-transfection, cells were lysed and subjected to luciferase activity assay, using the Dual-Glo Luciferase Assay System (Promega) on a LD400 Luminescence detector (Beckman Coulter, Fullerton, CA) as described (26). The results from three independent experiments with each run in duplicate or triplicate were expressed as fold induction after normalization with transfection efficiency and by arbitrarily setting the value of pGL3-Basic vector control as 1.

Identification of the transcription initiation site. To define the transcription initiation site of the mouse SAG, a PCR-based 5'-rapid amplification of cDNA ends (RACE) was done using the First-choice RLM-RACE kit with protocols suggested by the manufacturer (Ambion, Austin, TX) as described previously (26). Briefly, total mouse RNA was treated with calf intestine alkaline phosphatase to remove free 5'-phosphates from molecules of fragmented mRNA. The RNA was then treated with tobacco acid pyrophosphatase to remove the cap structure from full-length mRNA, leaving a 5'-monophosphate. A 5'-RACE adapter oligonucleotides, 5'-GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA-3', which is specific for the 5'-monophosphate, was ligated to the RNA population using T4 RNA ligase. A reverse transcription was done using random decamers, and the nest PCR then amplified the 5'-end of the mouse SAG mRNA using the 5'-RACE outer primer, 5'-GCTGATGGCGATGAATGAACACTG-3', or 5'-RACE inner primer, 5'-CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG-3' and a primer from the SAG mRNA, 5'-CAACACAGTCCTCTTGCTTGT-3'. The PCR products were cloned into pCR2.1 TA cloning vectors and insert fragments were sequenced. The transcription initiation site was determined by comparing the sequences immediately after the 5'-RACE adapter oligonucleotide to the mouse SAG gene sequence.

Gel retardation assay. Seven oligonucleotides and their corresponding complementary strands were synthesized (Invitrogen, Carlsbad, CA) as follows with the putative AP-1 binding sites in italics: Sag-AP1-1 (5'-GAAACTTGATTTGTAGACCA-3'), Sag-AP1-2 (5'-GCCTTATAACTCACAAGGAT-3'), Sag-AP1-3 (5'-GAGGGATGACTCAGTGGTTA-3'), Sag-AP1-3-MU (5'-GAGGGAAGACTCAGTGGTTA-3'), Sag-AP1-4 (5'-TTTGTTTGTTTGCTTGCTTGC-3'), Sag-AP1-5 (5'-AAGGTGTGAGCTACCACGCC-3'), Sag-AP1-6 (5'-CCTCGTGACGTCACTGGCGC-3'), Sag-AP1-6-Mu (5'-CCTCGAGACGTCACTGGCGC-3'), and Sag-AP1-7 (5'-GCGCCATCCAATCATCGCCGT-3'). The paired oligonucleotides were annealed and labeled with 32P using T4 polynucleotide kinase and [{gamma}-32P]ATP. Nuclear extracts were prepared from JB6-Cl.41 cells treated with TPA (20 ng/mL) for 16 h and subjected to gel retardation assay as described (27). In some experiments, the c-Jun antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or 50x excess of cold specific or nonspecific oligonucleotide was included to determine the binding specificity.

Chromatin immunoprecipitation assay. JB6-Cl.41 cells were left untreated or treated with TPA (20 ng/mL) for 12 h before being subjected to chromatin immunoprecipitation assay (ChIP) analysis, according to the protocol of Upstate Biotechnology, Inc. (Lake Placid, NY) as described (26, 28). Input samples (one tenth of original DNA lysates) or samples precipitated with no antibody (Ab–) or IgG for negative controls or c-Jun antibody (c-Jun) were PCR amplified. The primer sequences for the third AP-1 binding site (WT3) are mSAG-p-ChIP-3-01, 5'-GTTCTTGCCCTTCTGCTTACC-3', and mSAG-p-ChIP-3-02, 5'-TGCTGTGAGGTGTCAGATTC-3', to generate a 215-bp fragment. The primer sequences for the sixth AP-1 binding site are mSAG-p-ChIP-6-01, 5'-TGTAACTCCAGACAATGCTC-3', and mSAG-p-ChIP-6-02, 5'-AGCCAGACGGCGATGATTGG-3', to generate a 115-bp fragment.

Northern and Western blotting analyses. The cells were treated with TPA (20 ng/mL) for various periods up to 24 h and subjected to Northern analysis as described previously (29). For Western blotting analysis, cells were lysed in a Triton X-100 lysis buffer [20 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% Triton X-100, 5 mmol/L EGTA, and 5 mmol/L EDTA] with freshly added protease inhibitor tablet (Roche, Indianapolis, IN) for 30 min on ice followed by centrifugation for 30 min. Supernatants were measured for protein concentration using a Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA) and subjected to Western blotting (17) using antibodies against c-Jun, c-Fos (Santa Cruz Biotechnology), SAG, or ROC1/Rbx1. The ß-actin (Sigma, St. Louis, MO) was used as a loading control.

Lentivirus-based SAG overexpression in mouse JB6-Cl.41 cells. A lentivirus-based SAG-overexpressing construct (FG9-HA-SAG) was generated using lentivirus vector, FG9-EF1{alpha} (kindly provided by Dr. Colin Duckett, University of Michigan, Ann Arbor, MI). Following DNA sequencing confirmation, the HA-tagged SAG construct, along with the empty vector control, was transiently cotransfected into 293 cells with plasmids expressing gag, env, and polymerase. Virus-containing supernatants were collected and used to infect mouse JB6-Cl.41 cells. Expression of both HA-SAG and endogenous SAG was determined by Western blotting using anti-SAG antibody.

siRNA silencing. A lentivirus-based siRNA construct (H1, kindly provided by Dr. Ihor Lemischka, Princeton University, Princeton, NJ) was used to make an LT-SAG-siRNA. The sequences of SAG siRNA oligonucleotide are LT-bSAG01 (5'-AACAAGAGGACTGTGTTGTGGTCTGGTTCAAGAGACCAGACCACAACACAGTCCTCTTGTTTTTTGT-3') and LT-bSAG-02 (5'-CTAGACAAAAAACAAGAGGACTGTGTTGTGGTCTGGTCTCTTGAACCAGACCACAACACAGTCCTCTTGTT-3'). The control siRNA sequences are LT-Control-01 (5'-ATTGTATGCGATCGCAGACTTTTCAAGAGAAAGTCTGCGATCGCATACAATTTTTTGT-3') and LT-Control-02 (5'-CTAGACAAAAAATTGTATGCGATCGCAGACTTTCTCTTGAAAAGTCTGCGATCGCATACAAT-3'). The LT-SAG-siRNA plasmid, along with the control LT-Cont-siRNA, was confirmed by DNA sequence and then cotransfected into 293 cells, along with gag- and env-expressing plasmids. The supernatants containing viable LT-SAG-siRNA virus or LT-Cont virus were collected and used to infect JB6-Cl.41 cells.

To silence human SAG or ROC1/Rbx1 in 293 cells, a plasmid-based hairpin RNAs were made using the pU6pro vector (kindly provided by Dr. David L. Turner, University of Michigan; ref. 30) as described.1 The siRNA template oligonucleotides were made by Invitrogen. For psiSAG, the primer sequences are iSAG2-1 (5'-TTTGAACAAGAGGACTGTGTTGTGGTCTGGCAAGAGCCAGACCACAACACAGCCTGTTTTT-3') and iSAG2-2 (5'-CTAGAAAAACAAGAGGACTGTGTTGTGGTCTGGCCTCCAGACCACAACACAGTCCTCTTGTT-3'). For psi-hROC1, the primer sequences are iROC1-1 (5'-TTTGAAGACTTCTTCCATCAAGCTTCAAGAGAAGCTTGATGGAAGAAGCTTTT-3') and iROC1-2 (5'-CTAGAAAAAGACTTCTTCCATCAAGCTTCTCTTGAAGCTATGAAAAGTCTT-3'). The control psiCont sequences are iCont01 (5'-TTTGATTGTATGCGATCGCAGACTTCAAGAGAAGTCTGCGATCGCATACAATTTTT-3') and iCont02 (5'-CTAGAAAAATTGTATGCGATCGCAGACTTCTCTTGAAGTCTGCGATCGCATACAAT-3'). The cohesive ends of restriction enzymes BbsI (TTTG) and XbaI (CTAG) are in italics. The annealed synthetic primers were ligated into pU6pro vector predigested with BbsI and XbaI. The recombinant psiSAG plasmid was confirmed by DNA sequencing. To determine the effect of siRNA silencing of SAG or ROC1/Rbx1 on Fbw7-mediated c-Jun degradation, the 293 cells were transiently cotransfected with plasmids expressing ubiquitin and c-Jun with or without Fbw7 in the absence or presence of psi-hSAG or psi-hROC1. Thirty-eight hours post-transfection, cells were harvested and lysed. The cell lysates were then prepared and subjected to Western blotting using various antibodies.

In vivo ubiquitination. Human lung H1299 cells were transiently transfected with His-Ub and c-Jun alone or in combination with psiSAG or psiCont. Cells were harvested 48 h after transfection and split into two aliquots with one for direct Western blotting analysis and the other for in vivo ubiquitination assay as described (31). Briefly, cell pellets were lysed in buffer A [6 mol/L guanidinium-HCl, 0.1 mol/L Na2HPO4/NaH2PO4, 10 mmol/L Tris-HCl (pH 8.0), 10 mmol/L ß-mecaptoethanol] and incubated with Ni-NTA beads (Qiagen, Valencia, CA) at room temperature for 4 h. Beads were washed once with each of buffer A, buffer B [8 mol/L urea, 0.1 mol/L Na2HPO4/NaH2PO4, 10 mmol/L Tris-HCl (pH 8.0), 10 mmol/L ß-mecaptoethanol], and buffer C [8 mol/L urea, 0.1 mol/L Na2HPO4/NaH2PO4, 10 mmol/L Tris-HCl (pH 6.3), 10 mmol/L ß-mecaptoethanol]. Proteins were eluted from beads with buffer D [200 mmol/L imidazole, 0.15 mol/L Tris-HCl (pH 6.7), 30% glycerol, 0.72 mol/L ß-mecaptoethanol, 5% SDS]. The eluted proteins were analyzed by Western blotting for polyubiquitination of c-Jun with anti–c-Jun antibody.

Soft agar assay. A standard soft agar assay was done as detailed previously (32). Briefly, 10,000 of parental JB6-Cl.41 P+ cells or its Lenti-SAG transfectants (either SAG overexpressed or SAG silenced), along with the vector controls, were suspended in 0.33% agar containing 10% FCS in a 60-mm dish either in the absence (DMSO) or the presence of TPA (20 ng/mL). After grown in 37°C for 14 days, the cells were stained with p-iodonitrotetrazolium (1 mg/mL; Sigma) for overnight and the colonies with cell numbers more than eight were counted in five randomly selected areas from each dish. The results were from three independent assays, each run in duplicate. Statistical analysis was done using one-way ANOVA and Student's t test.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Molecular cloning and characterization of mouse SAG promoter. We have shown previously that SAG is inducible at the transcriptional level under redox-stress and hypoxic conditions (17, 33, 34). To determine the nature of SAG induction under various stress conditions, we went on and cloned a 1.3-kb genomic sequence upstream from the translational initiation site of mouse SAG gene (accession no. NT_039476). Bioinformatics analysis, using Transfac2 software, of this potential SAG promoter fragment revealed the consensus binding sites for many transcription factors including AP-1, nuclear factor-{kappa}B, p53, and hypoxia-inducible factor-1, among others. In this study, we characterized the regulation of the SAG promoter by AP-1. In this 1.3-kb potential SAG promoter fragment, there are seven putative AP-1 binding sites (Genbank accession no. DQ231571). Some are exactly matching the typical AP-1 consensus site 5'-TGAG/CTCA-3', whereas others are its derivatives identified by Transfac software as the potential AP-1 binding sites (data not shown). Before a detailed characterization of the SAG promoter, we first defined the transcription initiation site of the mouse SAG gene by the 5'-RACE experiment. DNA sequencing of several independent 5'-RACE clones all showed an adenine at –31 position upstream the translational initiation site at the very 5'-end of SAG transcript before reaching the adaptor sequence, indicating that it is the transcription initiation site.

AP-1 binds both in vitro and in vivo to the consensus binding sites in the promoter of mouse SAG gene. We next determined whether AP-1 directly binds to these putative consensus sites first in vitro by a gel retardation assay. Nuclear extracts were prepared from JB6-Cl.41 cells treated with TPA and incubated with 32P-labeled oligonucleotides, each containing a putative AP-1 binding site. Initial binding assay revealed that AP-1 only bound to two consensus sites, site 3 (TGACTCA) and site 6 (TGACGTCA) significantly, but not to other five putative sites (data not shown). To determine the binding specificity, these two sites were mutated by a single T->A substitution (for site 3, AGACTCA; for site 6, AGACGTCA). These mutant oligonucleotides, along with their WT controls, were subjected to gel retardation assay as shown in Fig. 1A . AP-1 bound significantly to the sites 3 (Fig. 1A, WT3, lane 1) and 6 (Fig. 1A, WT6, lane 7). These AP-1 bindings can be supershifted by an anti–c-Jun antibody (Fig. 1A, lanes 2 and 8), blocked by a specific cold oligonucleotide (Fig. 1A, lanes 3 and 9), but not by a nonspecific oligonucleotide (Fig. 1A, lanes 4 and 10). A single T->A point mutation completely abolished the AP-1 binding in both sites (Fig. 1A, lanes 5 and 6 and lanes 11 and 12). Thus, AP-1 specifically bound to two AP-1 sites in the mouse SAG promoter.


Figure 1
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  		Figure 1. AP-1 binds to two AP-1 consensus binding sites in the promoter of mouse SAG gene and transactivates mouse SAG promoter. A, in vitro AP-1 binding. Nuclear extracts were prepared from JB6-Cl.41 cells treated with TPA and incubated with 32P-labeled WT or mutant (MU) AP-1 consensus binding oligonucleotides. In some cases, anti–c-Jun antibody (Ab) was included to show a supershift, whereas, in other cases, 50x cold oligonucleotides (either WT or mutant) were included to show the binding specificity. Bands representing AP-1 binding and AP-1 + antibody binding. B, in vivo AP-1 binding. JB6-Cl.41 cells were left untreated or treated with TPA (20 ng/mL) for 12 h before being subjected to ChIP analysis. Input samples or samples precipitated with no antibody (Ab–), IgG, or c-Jun antibody (c-Jun) were PCR amplified using primers specific for the third AP-1 binding site (WT3, top) or the sixth AP-1 binding site (WT6, bottom), respectively. C and D, AP-1 site-dependent transactivation of the mouse SAG promoter. Left, representation of a series of luciferase reporters driven by various deletion or mutation mutants of mouse SAG promoter with putative AP-1 binding site boxed in black; right, the luciferase activity of each construct. HeLa cells were plated in 96-well plates and transiently cotransfected with luciferase reporters driven by the 1.3-kb SAG promoter fragment and its series of deletion or mutation mutants, along with the pGL-3–negative control, respectively. Renilla-luc reporter was included for normalization of transfection efficiency. Forty hours post-transfection, cells were lysed and subjected to luciferase activity assay. Luciferase activity was presented as fold activation from three independent transfections, each run in duplicate or triplicate after normalization of transfection efficiency by arbitrarily setting the value of pGL-3 as 1.

 
We further confirmed these AP-1 direct bindings using an in vivo ChIP. JB6-Cl.41 cells were left untreated or treated with TPA for 12 h to activate AP-1 and then subjected to ChIP analysis. As shown in Fig. 1B (top), a 215-bp PCR-amplified fragment, corresponding to the third AP-1 binding site (WT3), was detected in the input sample (Fig. 1B, top, lane 1) or in the samples immunoprecipitated with c-Jun antibody (Fig. 1B, top, lane 4) but not in two control samples either without antibody added (Fig. 1B, top, lane 2) or with IgG added (Fig. 1B, top, lane 3) for immunoprecipitation. This specific AP-1 binding was enhanced after TPA treatment (Fig. 1B, top, compare lanes 4 versus 8). Similarly, AP-1 bound to the sixth binding site (WT6), revealed as a 156-bp fragment in the input or in c-Jun antibody, immunoprecipitated samples (Fig. 1B, bottom, lanes 1 and 4) but not in other controls (Fig. 1B, bottom, lanes 2 and 3). The binding was again enhanced by TPA treatment (Fig. 1B, bottom). Thus, we conclude from these results that AP-1 indeed directly binds to two AP-1 consensus sequences in the promoter of the mouse SAG gene.

AP-1 binding site-dependent transactivation of mouse SAG promoter. We next determined the promoter activity of this 1.3-kb mouse SAG upstream fragment and its dependence of AP-1 binding sites. The luciferase reporters driven by the 1.3-kb fragment (7AP) and its series of mutants with AP-1 site either deleted or mutated were constructed and tested in a luciferase reporter assay. As shown in Fig. 1C, the construct (7AP) driven by this 1.3-kb fragment caused a 550-fold higher luciferase activity compared with the empty vector, indicating that this 1.3-kb fragment has a strong promoter activity. Deletion of 467 base nucleotides from the 5'-end (construct 5AP) caused a 1.6-fold reduction of the luciferase activity. Because this deleted fragment contained two putative AP-1 sites to which AP-1 failed to bind, it is likely that the cis-elements for other transcription factors rather than AP-1 contributed to this portion of the promoter activity. The deletion or mutation of the AP1-3 site (construct 4AP or Mu-5AP, respectively), which showed a strong AP-1 binding, caused an additional 1.4-fold reduction of the promoter activity, indicating that this AP-1 binding site alone contributed significantly to the promoter activity. Further deletion of 629 base nucleotides (construct 2AP), including two putative AP-1 sites to which AP-1 failed to bind, did not cause any change in luciferase activity, indicating that this fragment has no promoter activity. Further deletion or mutation of a strong AP-1 binding site 6 (construct 1AP or Mu-2AP, respectively) resulted in a 1.6-fold reduction of the promoter activity, indicating its strong contribution to the promoter activity. Moreover, the deletion of AP-1 binding site 7 (construct 0AP) caused an additional 1.4-fold reduction of the promoter activity, suggesting the contribution from other transcription factors. Finally, we directly compared the luciferase activity between two reporter constructs driven either by the 1.3-kb mouse SAG promoter (7AP) or by its mutant with a single mutation at both AP-1 binding sites 3 and 6 (Mu-3,6), which abolished the AP-1 binding. As shown in Fig. 1D, the mutations at these two AP-1 binding sites reduced the overall luciferase activity by 3.4-fold. Taken together, the results clearly showed that the promoter activity of mouse SAG gene is significantly dependent on two true AP-1 binding sites and strongly suggested an AP-1–dependent activation of the SAG promoter.

SAG induction by TPA. We next determined whether endogenous SAG mRNA and protein are subjected to induction by TPA, a typical tumor promoter and AP-1 activator. Mouse epidermal JB6-Cl.41 cells were treated with TPA (20 ng/mL) for various times and subjected to Northern analysis. As shown in Fig. 2A , SAG mRNA was detectable in JB6-Cl.41 cells and started to increase 1 h post-TPA treatment and reached a peak induction of 3.8-fold at 4 h. Increased mRNA started to decrease thereafter and returned toward the background level by 24 h. These results further indicated that SAG induction by TPA occurred at the transcriptional level. We next determined the SAG induction by TPA at the protein level. Cell lysates were prepared after TPA treatment for various periods and subjected to immunoblotting using anti-SAG antibody. As shown in Fig. 2B, similar to the pattern of SAG mRNA induction, the SAG protein levels started to increase at 1 h after treatment and reached a peak induction of up to 2.5-fold at 4 to 8 h. The induced SAG level then gradually reduced to the untreated control level at 24 h post-TPA treatment. We next determined the time course of c-Jun induction by TPA in this cell model. If SAG is a true target of c-Jun/AP-1, the time course of SAG induction should follow that of c-Jun induction. Indeed, the c-Jun levels increased 30 min after TPA exposure and continued to increase and reached a peak induction of 5-fold from 2 to 8 h. The induced levels started to decrease at 16 and 24 h (Fig. 2B). The results clearly showed that the time course of SAG induction by TPA closely followed that of c-Jun induction.


Figure 2
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  		Figure 2. SAG is subjected to TPA induction. A, induction of SAG mRNA expression by TPA in mouse JB6-Cl.41 cells. Subconfluent JB6-Cl.41 cells were subjected to TPA (20 ng/mL) treatment for indicated period times up to 24 h. Cells were harvested and subjected to total RNA isolation and Northern blot analysis using mSAG cDNA flanking the entire open reading frame as a probe. The membrane was stripped and reprobed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. The fold induction relative to the untreated control was shown after densitometry quantitation and GAPDH normalization. B, induction of SAG and c-Jun proteins by TPA in mouse JB6-Cl.41 cells. Subconfluent Cl.41 cells were subjected to TPA (20 ng/mL) treatment for indicated period times up to 24 h. Cells were harvested, lysed in Triton X-100 lysis buffer, and subjected (150 µg) to immunoblotting analysis using antibodies against SAG, c-Jun, and ß-actin. The fold induction relative to the untreated control was shown after densitometry quantitation and ß-actin normalization. C, induction of SAG protein in vector control and TAM67-overexpressed JB6-Cl.41 cells. Subconfluent cells were subjected to TPA (20 ng/mL) treatment for indicated period times up to 24 h. Cells were harvested, lysed in Triton X-100 lysis buffer, and subjected (150 µg) to immunoblotting analysis using antibodies against SAG, c-Jun, and ß-actin. The fold induction relative to the untreated control was shown after densitometry quantitation and ß-actin normalization. D, induction of c-Jun and SAG proteins by TPA in 293 cells. Subconfluent 293 cells were treated with TPA (10 ng/mL) for an indicated periods. Cells were harvested and subjected to Western blotting analysis using antibodies against c-Jun, SAG, and ß-actin. The numbers under each gel panel are fold changes after densitometry quantification with ß-actin normalization, setting the control value as 1.

 
To determine whether TPA-induced SAG induction requires c-Jun/AP-1, we used the JB6-Cl.41 cells overexpressing TAM67, a dominant-negative c-Jun that has been shown previously to block AP-1 activity in multiple cell models, including JB6-Cl.41 cells (24, 35–38). As shown in Fig. 2C, in vector-transfected control cells, TPA caused a gradual increase of SAG level with a peak induction of 3- to 3.5-fold at 4 to 8 h post-treatment. In contrast, TPA only slightly induced SAG expression up to 1.5-fold at 2 to 4 h post-TPA exposure in TAM67-transfected cells. Thus, the magnitude of TPA-induced SAG induction was significantly reduced when the AP-1 activity is blocked, indicating that SAG induction by TPA is largely AP-1 dependent. Finally, we extended our observation in JB6-Cl.41 cells that SAG is inducible by TPA to human 293 cells. As shown in Fig. 2D, the SAG level started to increase for ~2-fold at 2 to 4 h post-TPA exposure and reached the peak of a 3-fold increase at 16 h. The c-Jun expression at the basal level was hardly detectable in 293 cells but was rapidly induced by TPA, starting at 30 min for ~2-fold increase and reaching the peak of a 13-fold increase at 16 h. Again, the time course of SAG induction seemed to follow that of c-Jun induction. Taken together, the data from assays of gel retardation, luciferase-based transactivation, and endogenous induction in both mouse and human cell lines all suggested that SAG is inducible by TPA through AP-1 binding and transactivation and SAG is a novel AP-1 target gene.

SAG overexpression reduces both basal and TPA-induced c-Jun levels in JB6-Cl.41 cells. Few recent studies have shown that c-Jun is subjected to ubiquitination and degradation by SCF and DCX E3 ubiquitin ligases (22, 23). Because SAG is a RING component of SCF E3 ligase, we wondered whether SAG is involved in and if so, SAG would inhibit c-Jun levels on overexpression. We tested this hypothesis by using a lentivirus-based SAG construct expressing HA-tagged SAG. Infection of this construct into JB6-Cl.41 cells led to a 2.7-fold increase of SAG levels compared with the control vector (Fig. 3A ). This increased SAG level is within the physiologic range and is comparable with the level induced by TPA (see Fig. 2). These cells were then tested for both basal and TPA-induced c-Jun levels. In control cells, c-Jun was detectable and subjected to induction by TPA, starting at 1 h for 3-fold increase and reaching a peak of 6.4-fold increase at 8 h. In contrast, in SAG-infected cells, the basal c-Jun levels decreased ~2- to 3-fold and total TPA-induced levels were also reduced by 2-fold (Fig. 3B). As a negative control, SAG overexpression had no effects on p53, a protein not to be induced by TPA and not known to be a SAG substrate. The results suggested that SAG could reduce c-Jun levels, probably through promoting its degradation. To pursue this, we treated cells with MG132, a proteasome inhibitor, alone or in combination with TPA. If c-Jun is targeted for degradation by SAG, then MG132 should block it. Indeed, as shown in Fig. 3C, MG132 alone treatment caused a slight increase in basal level of c-Jun in control cells (Fig. 3C, lanes 1 versus 3) but a 2-fold increase in SAG-overexpressed cells (Fig. 3C, lanes 5 versus 7), resulting in similar c-Jun levels in both control and SAG-overexpressed cells (Fig. 3C, lanes 3 versus 7). Accordingly, SAG-mediated inhibition of c-Jun induction by TPA was also blocked by MG132 (Fig. 3C, compare lanes 2 versus 6 and lanes 4 versus 8).


Figure 3
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  		Figure 3. Reduction of both basal and TPA-induced c-Jun accumulations on SAG overexpression. A, SAG overexpression by lenti-HA-SAG infection. JB6-Cl.41 cells were infected with the empty lentivirus vector (LT-con) or lentivirus expressing HA-tagged SAG (LT-HA-SAG). Cell lysates were prepared and subjected to immunoblotting analysis using anti-SAG antibody and ß-actin as a loading control. Endo-SAG, endogenous SAG. B, SAG overexpression reduced both basal and TPA-induced c-Jun accumulation. Lentivirus-infected (empty vector control or HA-SAG) JB6-Cl.41 cells were treated with TPA for various periods and subjected to Western analysis using antibodies against c-Jun, p53, and ß-actin. The numbers under the gel panels are fold changes after densitometry quantification with ß-actin normalization, setting the value in LT-Con without TPA treatment as 1. C, MG132 blocked SAG-induced c-Jun reduction. Lentivirus-infected JB6-Cl.41 cells were treated with TPA or MG132 alone or in combination for 8 h and subjected to Western blotting using antibodies against c-Jun and ß-actin. The numbers under the gel panels are fold changes after densitometry quantification with ß-actin normalization, setting the value in LT-Con without TPA treatment as 1.

 
SAG silencing increases both the basal and TPA-induced c-Jun levels in JB6-Cl.41 cells. We further tested this hypothesis by using a lentivirus-based SAG siRNA silencing construct. Again, it is expected that SAG silencing should increase the levels of c-Jun, if it indeed promotes c-Jun degradation. As shown in Fig. 4A , lenti-SAG siRNA infection of JB6-Cl.41 cells reduced endogenous SAG levels by 80% compared with that of lenti-control siRNA infection, indicating that endogenous SAG was indeed significantly silenced. We then exposed these two lines to TPA for various periods of time up to 8 h followed by immunoblotting analysis for c-Jun protein levels. As shown in Fig. 4B, the basal level of c-Jun was very low but detectable in LT-Cont–infected cells. The levels of c-Jun continuously increased and reached a peak induction of 10-fold at 8 h post-TPA treatment. Significantly, in SAG silenced cells (LT-bSAGsi), the basal level of c-Jun was increased up to 3-fold than that in the control cells and TPA treatment further increased the c-Jun levels up to a total of 18-fold. Unlike c-Jun, SAG silencing had no effect on both basal and TPA-induced levels of c-Fos, an AP-1 component, but not a SAG substrate (Fig. 4C). Finally, SAG silencing had, once again, no effect on the levels of p53, which is neither inducible by TPA, nor a SAG substrate (Fig. 4C). These results strongly suggested that SAG is involved in down-regulation of both basal and TPA-induced levels of c-Jun, but not of c-Fos, in mouse JB6-Cl.41 cells.


Figure 4
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  		Figure 4. The accumulation of c-Jun levels on SAG siRNA silencing. A, reduction of SAG protein by siRNA silencing. JB6-Cl.41 cells were infected with lentivirus expressing either control siRNA (LT-CONsi) or siRNA targeting SAG (LT-bSAGsi). Cell lysates were prepared and subjected to immunoblotting analysis using anti-SAG antibody and ß-actin as a loading control. B and C, SAG silencing increased c-Jun levels in JB6-Cl.41 cells. Lentivirus-infected JB6-Cl.41 cells were treated with TPA for various periods and subjected to Western blotting analysis using antibodies against c-Jun, c-Fos, p53, and ß-actin. The numbers under the gel panels are fold changes after densitometry quantification with ß-actin normalization, setting the value in LT-CONsi without TPA treatment as 1.

 
SAG silencing inhibits c-Jun ubiquitination and blocks Fbw7-mediated c-Jun degradation. We next determined whether SAG siRNA silencing would inhibit c-Jun polyubiquitination by a commonly used in vivo ubiquitination assay (31). H1299 cells were transiently cotransfected with His-Ub and c-Jun, alone or in combination of plasmid expressing control siRNA or SAG siRNA. Ubiquitinated c-Jun was purified by Ni-NTA beads and detected by antibody against c-Jun. As shown in Fig. 5A , c-Jun polyubiquitination can be readily detected in cells transfected with His-Ub, c-Jun, and control siRNA (Fig. 5A, lane 4), indicating that endogenous SCF components are sufficient to promote c-Jun polyubiquitination on c-Jun overexpression via transfection. When cotransfected with plasmid that silenced SAG expression (Fig. 5A, middle), c-Jun polyubiquitination was remarkably inhibited (Fig. 5A, lane 3). Thus, SAG is required for c-Jun polyubiquitination.


Figure 5
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  		Figure 5. SAG silencing inhibited c-Jun polyubiquitination (A) and blocked Fbw7-mediated c-Jun degradation (B). A, H1299 cells were transiently transfected with His-Ub (lane 2) or c-Jun (lane 1) alone or His-Ub and c-Jun in combination with psiSAG (lane 3) or psiCon (lane 4) and subjected to in vivo ubiquitination assay by Ni-bead purification of ubiquitinated c-Jun followed by detection of polyubiquitinated c-Jun by c-Jun antibody. B, human kidney 293 cells were transiently transfected with ubiquitin, along with the pcDNA3 (lane 1); c-Jun and pcDNA3 (lane 2); c-Jun and Fbw7 (lane 3); c-Jun, Fbw7, and psi-hSAG (lane 4); c-Jun, Fbw7, and psi-hRbx1 (lane 5); or c-Jun, Fbw7, psi-hSAG, and psi-hRbx1 (lane 6). Forty hours post-transfection, cells were lysed and cell extracts were subjected to immunoblotting using antibodies against c-jun (top) and ß-actin as a loading control (bottom). The numbers under each gel panel are fold changes after densitometry quantification with ß-actin normalization, setting the value in (lane 1) as 1.

 
The F-box protein Fbw7 has been shown previously to promote c-Jun ubiquitination and degradation (22, 39). Because Fbw7 is the F-box component of SCF E3 ubiquitin ligase, it is likely that either SAG or ROC1/Rbx1, the RING component of SCF, would be required. We tested this hypothesis by determining whether silencing SAG or ROC1/Rbx1 by siRNA would block Fbw7-induced c-Jun degradation. Human 293 cells were transiently cotransfected with Ub, c-Jun, and plasmids expressing Fbw7, alone or in combination with psi-hSAG (to silence SAG) and/or psi-hRbx1 (to silence Rbx1/ROC1). As shown in Fig. 5B, endogenous c-Jun was hardly detectable (Fig. 5B, lane 1), whereas exogenously transfected c-Jun expressed well (Fig. 5B, lane 2). Consistent with a previous report (22), cotransfection of c-Jun with Fbw7 reduced the level of c-Jun by 5.6-fold (Fig. 5B, lanes 3 versus 2, from 1 to 0.18). Remarkably, cotransfection of Fbw7 with psi-hSAG, which caused a 2.5-fold reduction of endogenous SAG level (Fig. 5B, middle top, lanes 4 versus 1), reversed Fbw7-induced reduction of c-Jun from 5.6- to 1.3-fold (Fig. 5B, lane 4, from 1 to 0.18 and from 1 to 0.76). Similarly, cotransfection with psi-hROC1, which caused a 2-fold reduction of ROC1 (Fig. 5B, middle bottom, lanes 5 versus 1), also partially reversed Fbw7-induced c-Jun degradation from 5.5- to 1.6-fold (Fig. 5B, lane 5, from 1 to 0.18 and from 1 to 0.62). Simultaneously silencing both SAG and ROC1 (up to 2.5-fold of each) by cotransfecting both psi-hSAG and psi-hROC1 further inhibited c-Jun degradation by Fbw7 (Fig. 5B, lane 6). These results clearly showed that SAG as well as ROC1 is required for Fbw7-induced c-Jun degradation.

SAG expression inhibited TPA-induced soft agar colony formation in JB6-Cl.41 cells. It has been well established that c-Jun induction and AP-1 activation play a key role in TPA-induced tumor promotion (3, 5). Thus, by promoting c-Jun degradation, SAG might be able to inhibit tumor promotion activity of TPA. We tested this hypothesis by overexpressing SAG in JB6-Cl.41 cell line, a well-established line that is sensitive to TPA-induced neoplastic transformation as measured by anchorage-independent growth in soft agar (40, 41). The lentivirus expressing HA-tagged SAG was made and infected into JB6-Cl.41 cells to make stable SAG-expressing line, along with the vector control line. As shown in Fig. 6A , lenti-SAG stable JB6-Cl.41 cells expressed HA-tagged SAG at a level comparable with the endogenous SAG (Fig. 6A, right, lane 3 compared with lanes 1 and 2), indicating that lenti-SAG infection increased a total of SAG level by 2- to 3-fold. This is within the physiologic range because the endogenous SAG can be induced up to 3-fold after exposure to TPA (Fig. 2). All three JB6-Cl.41 lines (parental, vector infected, and HA-SAG infected) were subjected to soft agar assay to determine the effect of SAG expression on anchorage-independent growth (a measure of cellular transformation) in the absence or presence of TPA. As shown in Fig. 6A (left), all three lines had few background agar colonies in the absence of TPA. TPA treatment dramatically increased the number of agar colonies in parental cells, as expected. The vector virus infection caused a further increase of agar colonies, consistent to our previous observation made with the plasmid vector transfection (32). Significantly, exogenous SAG expression at the physiologic level reduced number of TPA-induced agar colonies by 40%, which is statistically significant (P < 0.05 compared with the parental control). Thus, a 2- to 3-fold increase of the SAG level could inhibit TPA-induced cellular transformation in mouse epidermal JB6-Cl.41 cells.


Figure 6
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  		Figure 6. Changes in SAG levels alter the anchorage-independent growth of JB6-Cl.41 cells. A, SAG overexpression reduced numbers of TPA-induced soft agar colonies. JB6-Cl.41 cells were infected with empty lentivirus vector or lentivirus expressing HA-tagged SAG and subjected to, along with parental JB6-Cl.41 cells, Western blotting analysis using anti-SAG antibody. ß-Actin was used as a loading control (right). All three lines (1 x 104 cells) were subjected to soft agar assay in the absence or presence of TPA (20 ng/mL) as described (32). Colonies (eight or more cells) were counted after 14 d. One-way ANOVA analysis (Origin 75 Demo, Origin Lab Corp., Northampton, MA) was done followed by Student's t test. *, P < 0.05, compared with parental cells (left). B, SAG siRNA silencing increased soft agar colony numbers in the absence or presence of TPA. JB6-Cl.41 cells were infected with lentivirus expressing scrambled control siRNA or SAG siRNA and subjected to, along with parental JB6-Cl.41 cells, Western blotting analysis using anti-SAG antibody. ß-Actin was used as a loading control (right). All three lines (1 x 104 cells) were subjected to soft agar assay in the absence or presence of TPA (20 ng/mL) as described (32). Colonies (eight or more cells) were counted after 14 d. One-way ANOVA analysis (Origin 75 Demo) was done followed by Student's t test. *, P < 0.05; ** P < 0.01, compared with parental cells, respectively (left).

 
SAG silencing increased the numbers of soft agar colonies in JB6-Cl.41 cells. Finally, we determined whether SAG siRNA silencing, which caused c-Jun accumulation at both basal and TPA-induced levels (Fig. 4), would promote cellular transformation as measured by this soft agar anchorage-independent assay in the absence or presence of TPA. As shown in Fig. 6B, lenti-SAG siRNA infection significantly reduced the level of endogenous SAG compared with scrambled control siRNA (Fig. 6B, right, lane 3 versus lanes 1 and 2). All these three lines were subjected to soft agar assay to determine the effect of SAG silencing on anchorage-independent growth in the absence or presence of TPA. As shown in Fig. 6B (left), in the absence of TPA, parental or control siRNA-infected cells formed few background agar colonies. However, SAG siRNA silencing caused a significant increase in soft agar colony numbers, suggesting that SAG silencing partially transformed this promoting sensitive line as a result of c-Jun accumulation. As expected, TPA treatment dramatically increased the number of soft agar colonies in parental cells as well as scrambled siRNA-infected cells. SAG silencing caused a further increase in soft agar colony numbers with statistically difference from both control cells. It is worthy noting that higher number of soft agar colonies was observed in both untreated and TPA-treated cells in this experiment compared with what was shown in Fig. 6A. This is due to the change of the serum lot number, a common phenomenon seen in soft agar assay with JB6-Cl.41 cells. Our results clearly showed that on SAG silencing to induce c-Jun accumulation, the tumor promotion-sensitive JB6-Cl.41 was partially transformed with a significant increase in its ability to grow in an anchorage-independent manner and that TPA treatment further enhanced this phenotype of cell transformation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown previously that SAG is a stress-responsive gene that is induced by redox agents and ischemia/reperfusion in multiple cell lines and normal tissues and acts as a cellular defense mechanism to protect cells or tissues from apoptosis induced by these stresses (16, 17, 33, 34). We report here that SAG is induced by TPA via AP-1–mediated transcriptional activation. Four lines of evidence presented in this study support notion that SAG is a direct AP-1 target. (a) Several AP-1 consensus elements were identified in the promoter of mouse SAG gene and AP-1 binds strongly to two of such elements in vitro and in vivo. (b) The SAG promoter activity is dependent of the true AP-1 binding sites. (c) Both SAG mRNA and protein are induced by TPA, a typical AP-1 activator, and the time course of SAG induction follows that of c-Jun induction. (d) SAG induction by TPA is c-Jun/AP-1 dependent. Targeting c-Jun by TAM67, a dominant-negative c-Jun mutant, significantly reduced TPA-induced SAG expression. Many AP-1 target genes have been identified previously that regulate cell proliferation, tumor promotion, and apoptosis (3, 5, 42). The unique feature for identification of SAG as yet another direct AP-1 target is that SAG is a component of SCF E3 ubiquitin ligase that on induction could in turn inhibit AP-1 activity through promoting c-Jun degradation, thus establishing a negative feedback loop that keeps AP-1 activity in check.

C-Jun was shown to be ubiquitinated and degraded by at least three types of E3 ubiquitin ligases, including SCF-Fbw7 (Skp1-cullin-Fbw7; refs. 22, 39), DCX (DDB1, cullin 4A, and X-box protein; ref. 23), and Itch (43, 44). ROC1/Rbx1, the RING component of SCF and DCX E3 ubiquitin ligases, was implicated, but not directly proven, in at least two c-Jun targeting E3s. Now, we showed here that SAG is indeed involved in c-Jun ubiquitination and degradation. When overexpressed, SAG remarkably reduced the levels of both basal and TPA-induced c-Jun, whereas on SAG silencing, both basal and TPA-induced levels of c-Jun were significantly increased. Furthermore, SAG silencing inhibited c-Jun polyubiquitination and Fbw7-induced degradation, indicating a requirement of SAG for c-Jun ubiquitination and degradation through SCF-Fbw7 E3 ubiquitin ligase. Our data also directly showed that like SAG, ROC1/Rbx1, which has the same tissue expression pattern as SAG (17, 45) but is expressed in a constitutive manner,3 was also involved in promoting c-Jun ubiquitination and degradation.

Mouse JB6 epidermal model consists of three variants with distinct transformation response phenotypes as judged by anchorage-independent growth in a soft agar colony formation assay on exposure to TPA: the variant with the promotion-resistant phenotype (P-) is resistant to TPA-induced transformation, as evident by its failure to form soft agar colonies in the presence of TPA. The variant with promotion-sensitive phenotype (P+), to which JB6-Cl.41 cells belong, is sensitive to TPA-induced transformation and will form agar colonies only in the presence of TPA. The third variant with transformed phenotype (Tx) will form agar colonies regardless of TPA. Thus, P-, P+, and Tx JB6 variants represent earlier-to-later stages of preneoplastic-to-neoplastic progression and provide an excellent cell model to study multistage carcinogenesis (41). It has been shown previously that in this JB6 model, the AP-1 activity was progressively elevated during preneoplastic-to-neoplastic progression (7) and blockage of TPA-induced AP-1 activity by a dominant-negative c-Jun (TAM67), encoding a transcriptionally inactive product, inhibited transformation induced by TPA and epidermal growth factor (8). This observation made in JB6 cell model has been extended to an in vivo transgenic mice model, in which TAM67 was overexpressed in mouse epidermal cells driven by a K14 promoter. Indeed, overexpression of TAM67 inhibits in vivo tumor formation induced by 7,12-dimethylbenz(a)anthracene (DMBA)/TPA chemical carcinogens as well as by UV exposure (35, 46). Likewise, we showed here that overexpression of SAG in physiologic level in mouse JB6-Cl.41 cells inhibited TPA-induced transformation, whereas SAG silencing induced cellular transformation of tumor promotion-sensitive JB6-Cl.41 cells. Furthermore, a SAG transgenic model, in which SAG expression was targeted to the epidermal cells by the K14 promoter (47), was generated and characterized. A decreased incidence with a longer latent period for the formation of papillomas induced by DMBA/TPA was also observed.4 Thus, AP-1 activation indeed plays a critical role in multistage carcinogenesis, and inactivation of AP-1 through targeting its component, such as c-Jun, would be a sound strategy for chemoprevention.

Our data presented here that (a) SAG is a direct AP-1 target, (b) SAG is involved in c-Jun degradation, and (c) SAG expression inhibits TPA-induced neoplastic transformation, establishing an autofeedback loop of c-Jun/AP-1-SAG, in analogue to p53-Mdm2 (48). TPA exposure induces the levels of c-Jun and c-Fos as well as c-Jun phosphorylation. Consequently, AP-1, consisting of Jun-Jun homodimer or Jun-Fos heterodimer, is activated to transactivate its target genes, leading to cell proliferation and tumor promotion (3, 5, 49, 50). Activated AP1 also transactivates SAG. On induction, SAG blocks AP1 effects through recruiting and complexing with SCF-Fbw7 E3 ligase to promote c-Jun ubiquitination and degradation. Induction of SAG or SAG-associated E3 ubiquitin ligases would therefore inhibit AP-1 activity, leading to inhibition of TPA-induced tumor promotion. Thus, SAG induction by TPA/AP-1 can serve as a cellular defensive mechanism to shut off cell proliferation and tumor promotion signals induced by prolonged AP-1 activation. It is worthy noting, however, that up-regulation of SAG by AP-1 will not only increase the activity of SAG-SCF-Fbw7 ligase to target c-Jun for degradation but may also induce the degradation of many other SAG-SCF ligase substrates, dependent on the availability of their specific F-box proteins. We therefore cannot exclude the possibility that degradation of other proteins may also contribute to SAG-mediated inhibition of neoplastic transformation. Nevertheless, our study suggests that SAG, on induction, inhibits TPA-induced neoplastic transformation and could serve as a cancer prevention target whose induction may help to delay the process of tumor promotion and progression through elimination of c-Jun and inactivation of AP-1.


   Acknowledgments
 
Grant support: National Cancer Institute grants 1R01CA111554, 1R01CA118762-01A1, and 1R21CA116982-01A1 and Charlotte Geyer Foundation (Y. Sun).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Ihor Lemischka for the H1 plasmid for lentivirus-based siRNA silencing, Dr. Colin Duckett for FG9 lentivirus used to make lenti-HA-SAG, Dr. David Turner for pU6pro vector used to make psiSAG and psiROC1, and Dr. Nancy Colburn (National Cancer Institute, Frederick, MD) for JB6-Cl.41 cells overexpressing TAM67.


   Footnotes
 
1 http://sitemaker.umich.edu/dlturner.vectors Back

2 http://www.gene-regulation.com/ Back

3 Unpublished data.. Back

4 Submitted for publication. Back

Received 10/30/06. Revised 12/24/06. Accepted 1/12/07.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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