Artigo Acesso aberto Revisado por pares

Stress response gene ATF3 is a target of c-myc in serum-induced cell proliferation

2005; Springer Nature; Volume: 24; Issue: 14 Linguagem: Inglês

10.1038/sj.emboj.7600742

ISSN

1460-2075

Autores

Kiyoshi Tamura, Bayin Hua, Susumu Adachi, Isil Guney, Junya Kawauchi, M. Morioka, Mimi Tamamori‐Adachi, Yujiro Tanaka, Yusaku Nakabeppu, Makoto Sunamori, John M. Sedivy, Shigetaka Kitajima,

Tópico(s)

Heat shock proteins research

Resumo

Article30 June 2005free access Stress response gene ATF3 is a target of c-myc in serum-induced cell proliferation Kiyoshi Tamura Kiyoshi Tamura Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Department of Cardiothoracic Surgery, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Bayin Hua Bayin Hua Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Susumu Adachi Susumu Adachi Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Isil Guney Isil Guney Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI, USA Search for more papers by this author Junya Kawauchi Junya Kawauchi Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Masaki Morioka Masaki Morioka Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Mimi Tamamori-Adachi Mimi Tamamori-Adachi Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Yujiro Tanaka Yujiro Tanaka Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Yusaku Nakabeppu Yusaku Nakabeppu Division of Neurofunctional Genomics, Department of Immunobiolgy and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Japan Search for more papers by this author Makoto Sunamori Makoto Sunamori Department of Cardiothoracic Surgery, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author John M Sedivy John M Sedivy Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI, USA Search for more papers by this author Shigetaka Kitajima Corresponding Author Shigetaka Kitajima Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Kiyoshi Tamura Kiyoshi Tamura Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Department of Cardiothoracic Surgery, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Bayin Hua Bayin Hua Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Susumu Adachi Susumu Adachi Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Isil Guney Isil Guney Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI, USA Search for more papers by this author Junya Kawauchi Junya Kawauchi Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Masaki Morioka Masaki Morioka Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Mimi Tamamori-Adachi Mimi Tamamori-Adachi Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Yujiro Tanaka Yujiro Tanaka Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Yusaku Nakabeppu Yusaku Nakabeppu Division of Neurofunctional Genomics, Department of Immunobiolgy and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Japan Search for more papers by this author Makoto Sunamori Makoto Sunamori Department of Cardiothoracic Surgery, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author John M Sedivy John M Sedivy Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI, USA Search for more papers by this author Shigetaka Kitajima Corresponding Author Shigetaka Kitajima Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Author Information Kiyoshi Tamura1,2, Bayin Hua1, Susumu Adachi3, Isil Guney4, Junya Kawauchi1, Masaki Morioka1, Mimi Tamamori-Adachi1, Yujiro Tanaka1, Yusaku Nakabeppu5, Makoto Sunamori2, John M Sedivy4 and Shigetaka Kitajima 1 1Department of Biochemical Genetics, Medical Research Institute and Laboratory of Genome Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan 2Department of Cardiothoracic Surgery, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan 3Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan 4Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI, USA 5Division of Neurofunctional Genomics, Department of Immunobiolgy and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Japan *Corresponding author. Department of Biochemical Genetics, Laboratory of Gene Structure and Regulation, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Tel.: +81 3 5803 5822; Fax +81 3 5803 0248; E-mail: [email protected] The EMBO Journal (2005)24:2590-2601https://doi.org/10.1038/sj.emboj.7600742 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The c-myc proto-oncogene encodes a transcription factor that promotes cell cycle progression and cell proliferation, and its deficiency results in severely retarded proliferation rates. The ATF3 stress response gene encodes a transcription factor that plays a role in determining cell fate under stress conditions. Its biological significance in the control of cell proliferation and its crosstalk regulation, however, are not well understood. Here, we report that the serum response of the ATF3 gene expression depends on c-myc gene and that the c-Myc complex at ATF/CREB site of the gene promoter plays a role in mediating the serum response. Intriguingly, ectopic expression of ATF3 promotes proliferation of c-myc-deficient cells, mostly by alleviating the impeded G1-phase progression observed in these cells, whereas ATF3 knockdown significantly suppresses proliferation of wild-type cells. Our study demonstrates that ATF3 is downstream of the c-Myc signaling pathway and plays a role in mediating the cell proliferation function of c-Myc. Our results provide a novel insight into the functional link of the stress response gene ATF3 and the proto-oncogene c-myc. Introduction The c-myc proto-oncogene regulates cell growth, proliferation, differentiation, and apoptosis, and its deregulation is implicated in the development of numerous human cancers (Henriksson and Luscher, 1996). Since c-Myc is a transcription factor and its expression is induced by serum and a variety of mitogens, it is important to identify its targets or the modulator gene(s) that mediate its effect on cell proliferation. Recently, several studies have used microarray analysis to compare Myc-driven changes in global gene expression. These studies have revealed that candidate c-Myc target genes fall into a broad spectrum of diverse functional categories, ranging from metabolic enzymes, biosynthesis of macromolecules such as RNA, protein and DNA, transcription, and cell signaling (Menssen and Hermeking, 2002; Fernandez et al, 2003; O'Connell et al, 2003). The target genes identified in these studies with respect to cell cycle control partially overlap but there are also non-overlapping outliers, perhaps stemming from the different assay systems used in controlling c-Myc expression. Thus, our knowledge of the functional link between c-myc's role in cell proliferation and those target genes is still limited. Homozygous deletion of the c-myc gene in mice leads to numerous developmental abnormalities and embryonic lethality at 10.5 days of gestation (Davis et al, 1993). Studies using conditional mutations or incremental reduction of c-Myc expression have shown that c-Myc is required to maintain the proliferation of embryonic fibroblasts, or to regulate the percentage of cells that re-enter the cell cycle (de Alboran et al, 2001; Trumpp et al, 2001). By contrast, a homozygous c-myc knockout in a rat fibroblast cell line is not lethal but results in a severely retarded cell growth phenotype, mainly due to lengthening of the G1 phase (Schorl and Sedivy, 2003). This culture model of c-myc knockout has been used extensively to investigate the roles of c-myc and its related target genes in the regulation of cell cycle progression. For instance, cyclin-dependent kinase 4 (CDK4) has been shown to partially restore the proliferation defect of c-myc-deficient cells (Hermeking et al, 2002). Effectors of the c-Myc signaling pathway and its crosstalk in cell proliferation, however, are still largely unknown. The stress-inducible transcription factor ATF3 is a member of the ATF/CREB family of basic leucine zipper (b-Zip) type transcription factors. It is induced upon exposure of cells to a variety of physiological and pathological stimuli (Hai et al, 1999, and references therein). The stress response of ATF3 is mediated through the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) or through a p53-dependent pathway, and stabilization of its mRNA is one mechanism for the control of the rapid induction of ATF3 in HeLa cells treated with anisomycin (Liang et al, 1996). This response is thought to have detrimental effects on the cell, such as cell cycle arrest and apoptosis (Yin et al, 1997; Hai et al, 1999; Cai et al, 2000; Kang et al, 2003). On the other hand, ATF3 is also rapidly induced in regenerating liver (Hsu et al, 1991), or in cells treated with growth-stimulating factors such as serum, epidermal growth factor, or fibroblast growth factor (Mohn et al, 1991; Iyer et al, 1999). ATF3 partially transforms chick embryo fibroblasts by promoting growth factor-independent proliferation (Perez et al, 2001), and induces DNA synthesis and expression of cyclin D1 in hepatocytes (Allan et al, 2001). Recently, we have reported that ATF3 supports cell survival by downregulating p53 transcription in endothelial and cardiac cells (Kawauchi et al, 2002; Nobori et al, 2002). ATF3 also protects neuronal cells from JNK-induced cell death by inducing heat shock protein 27 and Akt activation (Nakagomi et al, 2003). These data support a role for ATF3 in cell proliferation and survival. The transcriptional mechanism and biological significance of ATF3 expression in response to growth stimuli, however, remain elusive. Here we report that serum response of the ATF3 gene depends on c-myc gene status and that the retarded cell proliferation phenotype of c-myc-deficient cells is partially but significantly restored by ectopic expression of ATF3. This study demonstrates for the first time that ATF3 functionally interacts with the c-Myc signaling pathway, providing a novel insight into the functional coordination between cell cycle control and stress response. Results Serum induction of ATF3 is abrogated in c-myc-deficient cells ATF3 has been shown to be rapidly induced in response to partial hepatectomy in the rat (Hsu et al, 1991) or after serum treatment of HeLa cells and fibroblasts (Mohn et al, 1991; Iyer et al, 1999). We first serum-starved wild-type and c-myc-deficient HO15.19 rat fibroblasts for 48 h, and then stimulated them with serum. ATF3 mRNA was induced in wild-type cells, reaching a maximum level at 3 h after stimulation, whereas ATF3 protein was induced 3 h after stimulation with a peak at 6–12 h (Figure 1A). This rapid induction of ATF3 mRNA may partly be due to its increased stability, as reported (Liang et al, 1996). Under this condition, the c-myc mRNA and protein were more rapidly induced than ATF3, and reached their maximum levels at 1 and 3 h after serum treatment, respectively. In c-myc-deficient cells, on the other hand, ATF3 expression was almost completely abrogated at both the mRNA and protein levels (Figure 1A). ATF3 was induced in response to serum at levels similar to wild-type cells in both c-myc heterozygous cells and c-myc homozygous cells reconstituted with a c-myc transgene (Figure 1B), indicating that the serum induction of ATF3 gene depends on c-myc gene status. We next treated wild-type and c-myc-deficient cells with UV and etoposide, since ATF3 is also induced in response to various cytotoxic stimuli through JNK/MAPK pathway, or p53-dependent and -independent pathways (Yin et al, 1997; Hai et al, 1999; Cai et al, 2000). ATF3 was induced by these stress stimuli in both cell types at similar levels (Figure 1C), indicating that the signaling pathway(s) of ATF3 stress response to UV and etoposide is not c-Myc dependent. Figure 1.Serum induction of ATF3 in c-myc-deficient cells. (A) Upper panel: Northern blot analysis of ATF3 mRNA in serum-stimulated wild-type TGR1 and c-myc-deficient HO15.19 cells. Middle panel: RT–PCR analysis of ATF3 and c-Myc mRNA in wild-type cells after serum stimulation. Results are means with s.d. of triplicate experiments and shown as percent of the maximal amount of each transcript. Lower panel: Western blot analysis of ATF3 and c-Myc in wild-type and c-myc-deficient cells after serum stimulation. Immunoblot of tubulin is shown as a control of protein loading. (B) Western blot analysis of ATF3 and c-Myc in heterozygous c-myc-knockout +/− cells and homozygous c-myc-knockout −/− cells reconstituted with a c-myc transgene after serum stimulation. Cell extracts were prepared 12 h after serum treatment. (C) Stress-induced expression of ATF3 in wild-type and c-myc-deficient cells in response to UV (40 J/m2) or 20 μM etoposide (Eto). Control (cont) represents untreated cells. Download figure Download PowerPoint Serum induction of ATF3 is mediated by the ERK/MAPK pathway Serum treatment causes a rapid phosphorylation of extracellular signal-regulated kinase (ERK) and eventually leads to the activation of its target gene promoters. We thus examined the effect of PD98059, a specific MEK1 kinase inhibitor that suppresses the phosphorylation of ERK, on ATF3 induction. PD98059 almost completely abolished the serum induction of ATF3, whereas JNK inhibitor SP600125 or p38 inhibitor SB203580 had no significant effect. This indicates that the activation of MEK1/ERK is essential for the serum-induced expression of ATF3 (Figure 2A). Serum treatment caused rapid phosphorylation of ERK with similar kinetics in both wild-type and c-myc-deficient cells (Figure 2B), in agreement with a previous report (Mateyak et al, 1999). These data strongly suggest that serum induction of ATF3 is impaired downstream of the ERK/MAPK activation in c-myc-deficient cells. Figure 2.Serum-induced expression of ATF3 via ERK/MAPK signaling pathway. (A) Effect of MAPK inhibitors on serum-induced ATF3 expression in wild-type TGR1 cells determined by Western blot. The inhibitors were added 2 h before adding serum, and cell extracts were prepared 12 h after serum treatment. SP600125 and SB203580 were used at 10 and 25 μM, respectively. DMSO indicates vehicle only. (B) Phosphorylated ERK (P-ERK) and ERK (ERK) were measured at the indicated time after serum treatment of wild-type and c-myc-deficient cells by Western blot. Both 42 and 44 kDa isoforms were detected. In the lower panel, the activity 10 min after serum treatment in the presence of 25 μM PD98059 is shown. Download figure Download PowerPoint Serum induction of ATF3 promoter activity is suppressed in c-myc-deficient cells To determine whether the c-myc-dependent serum induction of ATF3 is regulated at the level of transcription, we performed an ATF3 gene reporter assay. Figure 3A illustrates the putative promoter elements of the ATF3 gene promoter, including c-Myc/Max binding sites as well as consensus motifs mediating the serum response (Liang et al, 1996), which are highly conserved between human and rat. As can be seen in Figure 3B, the ATF3 promoter was activated 3.8-fold in wild-type cells after serum treatment, and this activation was suppressed in the presence of PD98059. On the other hand, basal promoter activity in c-myc-deficient cells in the absence of serum was one-fifth the level of wild-type cells, and its induction by serum was significantly abrogated with only 1.1- to 1.3-fold activation. Both the basal and serum-induced promoter activities were fully restored in HO/myc3 cells containing the c-myc transgene. We next measured ATF3 promoter activity in wild-type cells using various deletion mutants. The deletion constructs down to −221 were all induced by serum (Figure 3C). In contrast, a further deletion down to −84 almost completely abolished serum induction. Since the region between −221 and −84 contains the putative ATF/CRE motif at −92 to −85, we performed the assay using the pLucATF3-1850m containing two point mutations at the ATF/CRE site (Cai et al, 2000). This mutation caused a marked reduction of serum-induced activity (Figure 3C). We next examined serum response of the reporter after expressing c-Myc protein in c-myc-deficient cells. c-Myc expression restored the serum response of pLucATF3-1850, -632, and -221, but had no effect on that of pLucATF3-84 and -1850m (Figure 3D), whereas control or LacZ expression had no effect. Combined, these results indicate that the ATF/CRE site in the ATF3 gene promoter represents one of the major elements responsible for the c-Myc-dependent serum response of ATF3. Figure 3.Repression of serum-induced ATF3 reporter activity in c-myc-deficient cells. (A) Human and rat ATF3 gene promoters with putative consensus elements for ATF/CREB, AP1, c-Myc/USF, and other factors are aligned (Liang et al, 1996). Human ATF3 reporter plasmids containing various deletions or two point mutations of ATF/CRE site are also depicted. (B) Serum induction of ATF3 promoter activity was assayed in wild-type TGR1 cells using pATF3Luc1850 in the presence or absence of 25 μM PD98059 for 24 h. Results are mean±s.d. of triplicate assays. Significant induction by serum compared with control, *P<0.05. (C) Wild-type cells were transfected with ATF3 reporters containing various deletions or ATF/CRE point mutations, and assayed for serum induction. Relative activity to that of minimal promoter pATF3Luc-84 was the mean with s.d. of triplicate experiments. Significant induction by serum compared with control, *P<0.05. (D) c-myc-deficient cells were transfected with various reporter plasmids along with empty, LacZ or c-Myc expression vectors and treated with 10% serum. Fold induction by serum is shown and represents the mean with s.d. of triplicate experiments. Significant induction by c-Myc compared with control, *P<0.05. Download figure Download PowerPoint ATF2/c-Jun complex binds the ATF/CRE motif and c-Myc is recruited to the proximal region of the ATF3 promoter We next performed a gel mobility shift assay using the ATF/CRE motif as a DNA probe. Incubation of probe with nuclear extracts from serum-starved cells produced bands over a broad size range (Figure 4A). These bands were specific for the ATF/CRE site, since assays in the presence of a wild-type cold probe but not mutant probe completely abolished their formation (data not shown; Cai et al, 2000). The intensity of these bands in serum-stimulated cells was significantly higher than in serum-starved cells. In supershift assays using various antibodies, both anti-ATF2 and anti-c-Jun antibodies resulted in supershifts, indicating that they are components of the protein–DNA complex (Figure 4A). Antibodies against ATF3, ATF4, CREB, JunB, or JunD did not result in any apparent supershifts (data not shown). Anti-c-Myc antibody decreased the intensity of the bands formed. The data indicate that ATF2 and c-Jun are induced to bind to the ATF/CRE motif in response to serum, and c-Myc might be involved in the complex formation (see also Supplementary Figure S1). To further investigate this possibility, we performed a chromatin immunoprecipitation (ChIP) assay to examine the in vivo recruitment of these factors to the ATF3 gene promoter. Both anti-ATF2 and anti-c-Jun antibodies immunoprecipitated the proximal region of the ATF3 gene promoter from −120 to +30, containing the ATF/CRE motif, in both serum-starved and serum-stimulated cells, whereas control IgG did not (Figure 4B). The anti-c-Myc antibody also immunoprecipitated the proximal promoter region after serum treatment, while it immunoprecipitated very little, if any, of this region in serum-starved cells. When ChIP assay was performed using an anti-ATF2 or an anti-c-Jun antibody, the promoter region was less efficiently immunoprecipitated in c-myc-deficient cells than in wild-type cells, indicating that ATF2/c-Jun binding is c-Myc dependent. In the light of these results, we conclude that ATF2 and c-Jun become activated to bind to the ATF/CRE motif in vivo, and c-Myc is also recruited to this region of ATF3 in response to serum. Figure 4.Binding of ATF2/c-Jun to ATF/CRE motif and c-Myc recruitment to the ATF3 gene promoter in response to serum. (A) Nuclear extract from wild-type cells serum starved or serum stimulated for 3 h was assayed for gel shift using radiolabeled DNA probe from −102 to −73 containing the ATF/CRE motif. From left: probe only, extract from the starved or serum-stimulated cells with or without the indicated antibodies. sp: specific bands; ns: nonspecific bands; arrow: supershift by anti-ATF2 antibody; arrowhead: supershift by anti-c-Jun antibody. In the inset, the expressions of c-Myc, ATF2, and c-Jun proteins were measured by Western blot. (B) Upper panel: Serum-starved or serum-stimulated wild-type cells were crosslinked with formaldehyde, and ChIP assay was performed using anti-c-Myc, anti-ATF2, or anti-c-Jun antibodies. ATF/CRE promoter region from −120 to +30, upstream region 1 from −370 to −120, and upstream region 2 from −570 to −370 were amplified by 26 cycles of PCR. Lower panel: c-myc-deficient cells were also subjected to ChIP assay, as above. ATF/CRE promoter region was also amplified by 28 cycles of PCR. (C) Whole-cell extracts from 293T cells overexpressing c-Myc, ATF2, and c-Jun were immunoprecipitated as in Materials and methods using anti-ATF2 (left panel), anti-c-Jun (middle panel), or anti-c-Myc antibody (right panel), respectively, and the resulting immune complex was subjected to Western blot analysis. Input was 10% of total. (D) Recombinant GST-c-Myc was mixed with ATF2 (upper left panel) or c-Jun alone (upper middle panel), or together (upper right panel), and GST pulldown assay was performed as in Materials and methods. In the lower panel, binding of GST-c-Myc with ATF2 or c-Jun was inversely assayed by immunoprecipitation using anti-ATF2 (lower left panel) or anti-c-Jun antibody (lower right panel), respectively. Input was 10% of total. Download figure Download PowerPoint c-Myc associates with ATF2/c-Jun complex both in vivo and in vitro To determine the nature of c-Myc recruitment to the proximal region of ATF3 gene promoter, we performed an immunoprecipitation assay using cells overexpressing c-Myc, ATF2, and c-Jun. As illustrated in Figure 4C (see also Supplementary Figure S2), both anti-ATF2 and anti-c-Jun antibodies specifically immunoprecipitated the ATF2/c-Jun complex. Under this condition, c-Myc protein was also co-precipitated by these antibodies, but not by control IgG. When the assay was performed with anti-c-Myc antibody, both ATF2 and c-Jun were co-precipitated, clearly indicating that c-Myc, ATF2, and c-Jun form a complex in vivo. To address whether c-Myc directly interacts with ATF2/c-Jun complex, an in vitro binding assay was performed using recombinant GST-c-Myc, ATF2, and c-Jun proteins. c-Myc was capable of binding to ATF2, while it formed very little, if any, complex with c-Jun (Figure 4D, left panel). This was also observed when the mixture was inversely immunoprecipitated with an anti-ATF2 or an anti-c-Jun antibody (Figure 4D, right panel). In contrast, when GST-c-Myc was mixed with ATF2 and c-Jun together, c-Myc bound to ATF2/c-Jun complex (Figure 4D, left lower panel). These data suggest that c-Myc forms a ternary complex with ATF2/c-Jun through its direct binding to ATF2. Expression of ATF3 promotes cell cycle progression in c-myc-deficient cells Homozygous deletion of the c-myc gene in rat fibroblasts significantly impedes G1-phase progression and results in the severe retardation of cell proliferation rates (Schorl and Sedivy, 2003). Since our data indicate that ATF3 is induced downstream of c-myc, we overexpressed the ATF3 protein in c-myc-deficient cells to test whether ATF3 can rescue their proliferation defect. We first employed adenovirus-mediated gene transfer to control the duration and amount of ATF3 expression such that it mimics the in vivo serum induction of ATF3. As illustrated in Figure 5A, ATF3 promoted the proliferation of c-myc-deficient cells, while LacZ expression had no effect. By contrast, ATF3 exerted only a marginal effect on the proliferation of wild-type cells. The level of ATF3 expression at 25 MOI was comparable to that observed in serum-induced wild-type cells while at 50 MOI it was two-to three-fold higher (Figure 5B), indicating that the expression of ATF3 at physiological amounts exerts cell proliferation activity. In the absence of serum, however, ATF3 was not capable of promoting cell proliferation. We also performed retrovirus gene transfers and established c-myc-deficient cell lines stably expressing ATF3 to examine the effect of ATF3 in a long-term assay. As in Figure 5C, these cell lines proliferated faster compared to their parent cells. Moreover, the A2 cell line expressing ATF3 at an amount higher than A1 displayed a higher proliferation rate. A1 cells displayed a roughly circular outline with occasional long processes as parental c-myc-deficient HO15 cells (Mateyak et al, 1997), demonstrating that the ATF3 expression causes no significant alteration of cell shape. Taken together, these results clearly indicate that ATF3 is capable of promoting the proliferation of c-myc-deficient cells. Figure 5.Proliferation of c-myc-deficient cells by ectopic expression of ATF3. (A) Wild-type and c-myc-deficient cells were infected with AdATF3 or AdLacZ at 25 MOI for 48 h and their proliferation rates were assayed. Relative cell number is shown as mean±s.d. of triplicate assays. (B) Dose-dependent cell proliferation by ATF3 in c-myc-deficient cells. Significant proliferation by ATF3 compared with control, *P<0.05. The lower panel shows the ATF3 expression in AdATF3-infected cells. In the left two lanes, the level of ATF3 expression in serum-stimulated wild-type cells is also shown. (C) Upper left panel: Proliferation rates of the c-myc-deficient cell lines A1 and A2 stably expressing ATF3. The relative cell numbers are the mean±s.d. of triplicate assays. Significant cell proliferation in A1 and A2 compared with control, *P<0.05. The upper right panel shows the ATF3 expression in A1 and A2 cells. Lower panel: Morphology of wild-type, c-myc-deficient, and A1 cells is shown. Scale bar, 20 μm. Download figure Download PowerPoint Cell cycle promoters are upregulated and G1-phase progression defect is alleviated by ATF3 in c-myc-deficient cells To assess the effect of ATF3 on cell cycle progression, we measured Cdk4 and Cdk2 activities in

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