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J. Biol. Chem., Vol. 276, Issue 29, 27272-27280, July 20, 2001

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Activating Transcription Factor 3 Induces DNA Synthesis and Expression of Cyclin D1 in Hepatocytes^*

Alison L. Allan^§, Chris Albanese^¶, Richard G. Pestell^¶, and Jonathan LaMarre

From the  Department of Biomedical Sciences, University of Guelph, Guelph, Ontario N1G 2W1, Canada and the ^¶ Albert Einstein Comprehensive Cancer Center, Division of Hormone-dependent Tumor Biology, Albert Einstein College of Medicine, Bronx, New York 10461

Received for publication, April 10, 2001, and in revised form, May 23, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Activating transcription factor 3 (ATF3) is an early response gene that is induced rapidly during in vivo situations of cellular growth such as liver regeneration. However, neither the physiological function nor the potential target genes of this transcription factor related to cellular proliferation have been identified in the liver or other tissues. We demonstrate here that endogenous ATF3 mRNA expression is rapidly induced up to 4-fold upon mitogenic stimulation of quiescent Hepa 1-6 mouse hepatoma cells. Overexpression of exogenous ATF3 results in a significant, dose-dependent increase in DNA synthesis of up to 140% over control cells. ATF3-transfected cells also display significantly higher rates of [³H]thymidine incorporation in comparison with nontransfected controls in the presence of serum. Northern blot analysis and co-transfection experiments demonstrate that overexpression of ATF3 enhances cyclin D1 mRNA expression and activates the cyclin D1 promoter 2.5-fold when activating protein-1 (AP-1) and cyclic AMP response element (CRE) sites within the promoter are intact. ATF3-mediated promoter activation is reduced to 1.3-fold and 1.6-fold respectively when the AP-1 or CRE sites are mutated, and mutation of both sites simultaneously leads to the complete abrogation of promoter activation. Furthermore, DNA-binding studies demonstrate that ATF3 binds directly to the AP-1 site within the cyclin D1 promoter. These results indicate that ATF3 expression stimulates hepatocellular proliferation, suggesting that this effect is mediated, at least in part, by the ATF3-dependent activation of cyclin D1 transcription.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The mammalian liver has a remarkable capacity to regenerate after the loss of hepatic tissue due to surgical, viral, traumatic, or toxicological injury (1, 2). As liver cells exit their normally quiescent state (G₀ of the cell cycle), a group of immediate-early genes are rapidly induced that aid in the G₀/G₁ transition (3-5). Among these genes are many of the proto-oncogenes implicated in cancer growth. Also induced during this immediate-early response is activating transcription factor 3 (ATF3).¹

ATF3 encodes a 21-kDa leucine zipper transcription factor that shows high sequence homology to the murine transcription factor LRG-21 (6) and to LRF-1 (rat liver regeneration factor-1) (7). ATF3 is expressed at very low levels in normal, quiescent cells but can be rapidly and highly induced in different cell types by multiple and diverse extracellular signals including mitogens (serum, epidermal growth factor) (8), cytokines (interferon , interleukin-4) (6), and genotoxic agents (ionizing radiation, UV light) (9). In vivo, ATF3 is highly expressed in situations of cellular growth or stress, such as liver regeneration, brain seizure, ischemia-reperfusion of the heart, or nerve damage (7, 10-12).

ATF3 contains the basic region and leucine zipper (bZIP) motif characteristic of the bZIP superfamily of transcription factors, which includes members of the CCAAT/enhancer-binding protein (C/EBP) family, the Jun/Fos (AP-1) family, and the ATF/CREB family (of which ATF3 is a member) (7, 13). ATF3 can homodimerize, but it preferentially heterodimerizes with c-Jun, JunB, JunD, ATF2, and gadd153/CHOP10 (growth arrest/DNA damage C/EBP homologous protein) to facilitate DNA binding to AP-1 or ATF/CRE consensus sites. Depending on dimerization partner, target promoter, and cellular context, ATF3 can act either as a transcriptional activator or repressor (7, 13-16).

Relatively little is known about the regulation or biological importance of ATF3 induction. Although most ATF3 studies to date have focused on DNA binding and heterodimer formation during cellular stress responses (Ref. 9 and references therein), a few studies have shown increased ATF3 expression in cancer cell lines (17, 18) and an interaction between ATF3 and the X protein of the hepatitis B virus (19, 20). X protein has been implicated in the development of human hepatocellular carcinoma during chronic hepatitis B virus infection via transactivation of viral and cellular transcription (21). Furthermore, ATF3 expression has been shown to be elevated during the promotion and progression stages of the in vivo resistant hepatocyte model of liver carcinogenesis (22). Therefore, although the biological functions of ATF3 have yet to be elucidated, there is strong circumstantial evidence that this transcription factor plays an important role in the regulation of normal and neoplastic hepatocyte growth responses.

The expression of ATF3 and other immediate-early genes is followed by the sequential expression of a set of delayed-early genes and the onset of DNA synthesis (23). This ordered progression through the cell cycle is controlled to a large extent by the cyclin-dependent kinases and their specific regulatory subunits, the cyclin proteins (24-26). A number of specific cyclins have been isolated and characterized in mammalian cells, and their temporal patterns of expression have been mapped to specific phases of the cell cycle. For example, cyclins A, B1, and B2 demonstrate maximal expression relatively late in the cell cycle and are thought to regulate the cell's transition to mitosis, whereas cyclins C, D1, D2, D3, and E reach peak expression during the G₁ phase and appear to drive cells toward the G₁/S transition (24, 25, 27).

Previous studies have demonstrated an up-regulation of cyclin D1 during the G₁ phase of the hepatic cell cycle following partial hepatectomy (28, 29) or mitogen stimulation of cultured hepatocytes (30), as well as during the continuous in vivo liver regeneration that occurs in chronic liver diseases such as hepatitis and cirrhosis (29). Furthermore, amplification and/or overexpression of cyclin D1 has been implicated in many human cancers, including hepatocellular carcinogenesis (31-35), suggesting that cyclin D1 may be a critical mediator of the cell cycle in the liver and elsewhere.

In the present study, we have utilized the Hepa 1-6 mouse hepatoma cell line (36) to characterize the expression and biological effects of ATF3 during in vitro hepatic cell growth. We demonstrate that ATF3 is rapidly and highly induced in quiescent Hepa 1-6 cells following treatment with serum or hepatic mitogens. Based on these results and the findings of previous studies (7, 8), we hypothesized that ATF3 is an important regulator of hepatocyte proliferation and that overexpression of ATF3 would lead to increased cellular growth via transcriptional activation of downstream cell cycle mediators. In the search for potential molecular targets of ATF3, cyclin D1 represented a logical possibility for several reasons. Cyclin D1 exhibits delayed-early expression kinetics in the liver, peaking mid-to-late G₁ (3, 5, 29), which indicates that, from a temporal perspective, the pattern of cyclin D1 expression is consistent with that of an ATF3 target. More importantly, the cyclin D1 promoter contains two of the consensus binding sites for ATF3: an AP-1 (TGAGTCAG) site at 953 and an ATF/CRE (TAACGTCA) at 54 (37-39). Finally, the cyclin D1 promoter has previously been shown to be activated by other members of the bZIP superfamily of transcription factors, including c-Jun (38), c-Fos, FosB (39), and ATF2 (40). We report here a novel role for ATF3 during hepatocyte proliferation and suggest that ATF3 regulates hepatocyte growth in vitro by activating the cyclin D1 promoter.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- The Hepa 1-6 mouse hepatoma cell line was obtained from American Type Culture Collection (Manassas, VA). Hepa 1-6 cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/liter glucose, 10% fetal bovine serum (FBS), penicillin (50 units/ml), and streptomycin (50 µg/ml), at 37 °C, 5% CO₂. Cells were plated onto culture dishes at a density of 2.5 × 10⁶ cells/60-mm dish (RNA studies), 7.0 × 10⁶ cells/100-mm dish (protein studies), 1.0 × 10⁵ cells/well in 24-well plates (DNA synthesis studies), and 4.0 × 10⁵ cells/well in 6-well plates (reporter gene studies). All media, sera, and supplements were from Life Technologies, Inc. All tissue culture plastics were from Sarstedt Inc., St. Leonard, QC, Canada.

RNA isolation and Northern Blot Analysis-- Cells were cultured as described above. 24 h after plating, cells were serum-starved for a further 36 h to synchronize cultures into quiescence. Following serum starvation, cells were cultured in the presence or absence of FBS (10%), epidermal growth factor (EGF) (10 ng/ml), or hepatocyte growth factor (HGF) (20 ng/ml). At specified times, cells were washed once in cold PBS and scraped directly into Trizol^TM reagent (Life Technologies, Inc.). Total cellular RNA was isolated per the manufacturer's recommendations. Total RNA (15 µg) from each time point was DNase-treated (DNA-free^TM, Ambion, Austin, TX) before being subjected to electrophoresis in 0.8% formaldehyde/agarose gels and transferred to nylon membranes (Hybond N; Amersham Pharmacia Biotech) by capillary transfer (48 h). Membranes were UV-cross-linked and prehybridized at 42 °C for 1 h in 6 × SSPE (saline/sodium phosphate/EDTA), 0.5% SDS, 5 × Denhardts' reagent, 20 µg/ml salmon sperm DNA, and 50% formamide. cDNA probes for murine ATF3 (6) (kindly provided by Dr. B. Drysdale, Veterans Affairs Medical Center, Baltimore, MD) and cyclin D1 (ATCC) were radiolabeled using 50 µCi of [-³²P]dCTP and the Rediprime^® random primer labeling system (Amersham Pharmacia Biotech). The radiolabeled cDNA probes were incubated with the membranes at 42 °C for 18 h in a solution identical to the prehybridization solution. Membranes were subjected to two low stringency washes (15 min each) in 2 × SSC, 1% SDS at 42 °C and one high stringency wash (15 min) in 0.1× SSC, 0.1% SDS at 55 °C (cyclin D1) and 60 °C (ATF3). Northern blots were analyzed using a Molecular GS250 Imager (Bio-Rad) located in the Clarice Chalmers Molecular Imaging Facility, Biomedical Sciences, University of Guelph. To normalize RNA load, membranes were rehybridized with a radiolabeled cDNA probe for murine 7 S RNA (Dr. A. Balmain, Onyx Pharmaceuticals, Richmond, CA). Band intensity was quantified with the Molecular Analyst (Bio-Rad) software program and expressed as a percentage of the level at quiescence.

Plasmids-- A 1010-base pair EcoRI/BstEII cDNA fragment representing the entire coding region of murine ATF3 (6) was cloned into the pcDNA3.1/Zeo⁺ expression plasmid (Invitrogen, Carlsbad, CA) using standard cloning techniques (41) (Fig. 2). The cyclin D1 promoter/reporter constructs 964CD1LUC, 964AP-1mutCD1LUC, 1745CD1LUC, and 1745CREmutCD1LUC were constructed as described previously using the pA₃LUC vector (38), and the 1745AP-1/ CREmutCD1LUC was generated by polymerase chain reaction-directed mutagenesis to mutate both the AP-1 site at 953 and the CRE site at 54 (Fig. 6A). The cytomegalovirus (CMV Sport -gal^TM, Life Technologies, Inc.) reporter plasmid (0.150 µg/ml) was co-transfected and used as a control for transfection efficiency in all transfection experiments. The fidelity of all plasmids was determined using restriction mapping and confirmed by dye terminator cycle sequencing using an ABI Prism 377 DNA automated sequencer at the Molecular Supercentre, University of Guelph.

Transfection-- Cells were cultured as described above. 24 h after plating, cells were serum-starved for a further 36 h to synchronize cultures into quiescence. Following serum starvation, cells were transfected using LipofectAMINE^TM reagent (Life Technologies, Inc.) per the manufacturer's instructions. Briefly, DNA-liposome complexes were formed by incubating 0.05-1.5 µg/ml plasmid DNA and 12 µg/ml LipofectAMINE^TM for 30 min in serum-free DMEM. The DNA-liposome complexes were then added dropwise to each culture dish and incubated at 37 °C, 5% CO₂ for 12 h. Following transfection, cells were maintained in culture in serum-free DMEM. Transfection conditions for Hepa 1-6 cells were previously optimized to yield an average transfection efficiency of ~50% (data not shown).

Western Blot Analysis-- Cells were cultured and transfected as described above. 48 h after transfection, cells were washed twice in cold PBS and harvested in radioimmune precititation buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in 1× PBS) supplemented with 100 µg/ml aprotinin, 100 µg/ml phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate. Whole cell lysates (30 µg) for each sample were subjected to electrophoresis in 12% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Immobilon^TM, Millipore, Bedford, MA) by electrophoretic transfer at 300 mA for 3 h. After transfer, gels were stained with Coomassie Blue to confirm transfer efficiency. Membranes were incubated for 18 h in a blocking solution (5% skim milk, 0.05% Tween 20). After blocking, the abundance of ATF3 protein was quantified using a rabbit polyclonal IgG antibody for ATF3 (1:500, Santa Cruz Biotechnologies, Santa Cruz, CA) for 1 h, washed (4 times) with the blocking solution, and then incubated (1 h) with a biotin-conjugated monoclonal anti-rabbit IgG antibody (1:10000, Sigma). ATF3 protein was visualized using an enhanced chemiluminescence system (Roche Molecular Biochemicals).

Determination of DNA Synthesis-- 48 h after transfection, cells were cultured in the presence or absence of FBS (0-20%) for 24 h and then labeled with 0.5 µCi/ml [methyl-³H]thymidine (1.3 TBq/mmol; ICN, Aurora, OH) for 4 h. Radioactive medium was removed, cells were detached using 250 µl of cell lysis buffer (0.5% SDS, 100 mM NaOH), rocked gently for 1 h, and harvested into 5 ml of liquid scintillation fluid (Ecolite, ICN). [ ³H]thymidine incorporation was assessed using a Tri Carb 2900TR liquid scintillation analyzer (Packard) and expressed as a percentage of control ([ ³H]thymidine incorporation in nontransfected cells). Data were presented as the mean ± S.E. of the mean (n = 15). Paired samples were compared by paired t test, and values of p < 0.05 were considered significant.

Luciferase and -Galactosidase Reporter Assays-- 48 h after transfection, cells were washed twice with cold PBS. Cells were harvested with 150 µl of 1× reporter lysis buffer (Promega, Madison, WI). Cells were snap-frozen on dry ice, thawed, vortexed for 15 s, and centrifuged at 13,000 rpm for 1 min. Cell extracts were collected and stored at 70 °C. To measure luciferase activity, 100 µl of luciferase assay reagent (Promega) was added to 20 µl of diluted (1:10) cell extract. Each sample was vortexed for 3 s, and the light intensity was measured for 10 s in a Turner Luminometer 20e (Turner Designs, Sunnyvale, CA). To measure -galactosidase activity, 50 µl of diluted (1:10) cell extract was added to 50 µl of 2× assay buffer (Promega) in a 96-well microtiter plate and incubated at 37 °C for 30 min. The reaction was stopped by adding 150 µl of 1 M sodium bicarbonate (Promega). -Galactosidase activity was measured at 410 nm using a microplate autoreader (Titertek Multiskan^® MCC/340, Flow Laboratories, Mississauga, Ontario, Canada). After adjusting for the dilution factor, luciferase values for each sample were normalized to the -galactosidase activity and expressed as a percentage of control (luciferase activity in cells transfected in the absence of the pcDNA3.1/ATF3 expression plasmid). Data were presented as the mean ± S.E. of the mean (n = 15). Paired samples were compared by paired t test, and values of p < 0.05 were considered significant.

Preparation of Nuclear Extracts-- Cells were washed twice with cold PBS, harvested in 1 ml of PBS, pelleted, and resuspended in 3 volumes of Buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT). The cells were disrupted with 20 up-and-down strokes with a type B Dounce homogenizer. The homogenate was centrifuged at 4000 rpm for 15 min to isolate crude nuclei. The crude nuclei were resuspended in 1 volume of Buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl₂, 600 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT). The nuclei were extracted for 30 min with continuous gentle mixing and then centrifuged at 13,000 rpm for 30 min. The resulting nuclear extracts were collected and stored at 70 °C.

DNA Binding Studies-- Electrophoretic mobility shift assays (EMSA) were performed as described previously (6, 7). Briefly, pre-annealed high pressure liquid chromatography-purified double-stranded oligonucleotides corresponding to the AP-1 (5'-AAAAAATGAGTCAGAATGGAGATC-3') and CRE (5'-ACAACAGTAACGTCACACGGACTA-3') sites in the cyclin D1 promoter were end-labeled with [-³²P]ATP (Amersham Pharmacia Biotech) using T4 polynucleotide kinase (Promega) (underlines indicate consensus sequences). Radiolabeled oligonucleotides (25,000 cpm) were mixed with 1.5 µg of nuclear extract from nontransfected, serum-stimulated cells or ATF3-transfected cells and incubated in binding buffer (10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 5% (v/v) glycerol) at 30 °C for 30 min. Samples were electrophoresed in a nondenaturing 5% polyacrylamide gel with Tris-glycine buffer. DNA-protein complexes were visualized by autoradiography.

To determine whether ATF3 represented the specific DNA-binding protein identified by the EMSA, a DNA-binding assay utilizing biotinylated oligonucleotides and streptavidin-agarose was performed as described previously (42). The binding reaction was carried out as described above using 30-50 µg of nuclear extract and 0.6 µg/ml biotinylated AP-1 and CRE oligonucleotides identical in sequence to those used for the EMSA. Streptavidin-agarose (Life Technologies, Inc.) was treated with 1 mg/ml BSA and 0.2 mg/ml salmon sperm DNA to block nonspecific binding, diluted 1:5, and equilibrated for 1 h in binding buffer. The pretreated streptavidin-agarose was subsequently added to the DNA-protein complexes and incubated for a further 30 min at room temperature with continuous mixing. The streptavidin-agarose was washed (3 times) with 1 ml of binding buffer. Proteins bound to biotin-target DNA were collected after a 1-h incubation in 1% SDS, 10 mM DTT and analyzed by Western blot analysis as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ATF3 Is Rapidly and Highly Induced in Mitogen-stimulated Hepa 1-6 Cells-- In an attempt to mimic the resting G₀ state of the normal in vivo liver, cultured cells were synchronized into quiescence by serum deprivation before mitogenic stimulation. Endogenous ATF3 mRNA was expressed at very low levels in quiescent cells but was rapidly and highly induced as early as 0.5 h following treatment with 10% FBS (Fig. 1A). Densitometric analysis (Fig. 1B) confirmed that ATF3 expression showed peak induction 1 h after serum stimulation and rapidly dropped off to below base-line levels by 4 h. Furthermore, treatment of quiescent Hepa 1-6 cells with either HGF (20 ng/ml) or EGF (10 ng/ml) resulted in a similar trend of ATF3 mRNA induction (Fig. 1C), although the magnitude of ATF3 induction by HGF was slightly higher than that induced by EGF (Fig. 1D). Again, peak expression occurred 1 h after growth factor stimulation and then rapidly returned to base-line levels.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Effect of serum stimulation (A and B) and growth factor stimulation (C and D) on endogenous ATF3 mRNA expression in quiescent Hepa 1-6 mouse hepatoma cells. Cells were incubated in serum-free DMEM (36 h) before addition of FBS (10%), HGF (20 ng/ml), or EGF (10 ng/ml) to the medium. 15 µg of total RNA was isolated for each time point and assessed by Northern blot analysis with a murine cDNA probe for ATF3. Load normalization was verified by reprobing blots with a 7 S RNA cDNA probe. Panels A and C show representative Northern blots. In panel C, quiescent cells stimulated with 10% FBS for 1 h (Pos) were included as a positive control. Panels B and D show the relative intensity of endogenous ATF3 expression 0-24 h following serum stimulation and 0-4 h following growth factor stimulation, respectively. Band intensities of three independent experiments were quantified densitometrically and expressed as a percentage of control (time 0, quiescence).

Overexpression of ATF3 Stimulates Hepatocyte DNA Synthesis in Vitro-- Characteristic of the transition from hepatic quiescence to proliferation is the increased expression of a set of immediate-early genes (3-5). It remains unclear whether these genes act in concert, each with a specific role, or whether all of these genes act in a similar manner and instead represent a sort of molecular "safety network" such that expression of a specific gene alone is actually sufficient to drive cells through the G₀/G₁ transition. The very rapid up-regulation of ATF3 following mitogen stimulation of Hepa 1-6 cells suggests that this gene may be an important mediator of hepatic cell growth. To determine whether ATF3 expression was sufficient to induce cellular proliferation in vitro, we cloned a DNA fragment containing the entire coding region of the ATF3 gene into the pcDNA3.1/Zeo⁺ expression vector (Fig. 2) for the purpose of transfecting this plasmid into Hepa 1-6 cells and measuring the resultant effect on DNA synthesis. Fig. 3A illustrates that cells transfected with the control plasmid (pcDNA3.1; empty expression vector) showed no significant change in DNA synthesis. However, transient transfection of quiescent Hepa 1-6 cells with increasing concentrations or "doses" of pcDNA3.1/ATF3 resulted in a dose-dependent increase in DNA synthesis activity relative to nontransfected cells. At all doses of pcDNA3.1/ATF3, DNA synthesis was significantly higher than control, and there were significant differences between each dose (Fig. 3A).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Schematic representation of the pcDNA3.1/ATF3 expression vector. A 1010-base pair EcoRI/BstEII cDNA fragment containing the entire coding region of murine ATF3 was cloned into the pcDNA3.1/Zeo+ expression plasmid. The cDNA fragment includes the initial methionine codon (ATG) ~170 base pairs from the 5'-end and the internal stop codon (TAA) ~713 base pairs from the 5'-end. The cytomegalovirus Sport -galTM promoter/enhancer drives efficient high level expression of ATF3. The bovine growth hormone polyadenylation (BGH pA) signal facilitates efficient transcriptional termination, polyadenylation, and enhanced stability of the ATF3 mRNA. ori, origin of replication.

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Dose-dependent effects of exogenous ATF3 overexpression on [3H]thymidine incorporation (A), mRNA levels (B), and protein levels (C) in quiescent Hepa 1-6 cells. Cells were incubated in serum-free DMEM (36 h) before liposome-mediated transfection (12 h) with 0, 0.25, 0.5, or 1.5 µg/ml pcDNA3.1/ATF3 expression plasmid. In all experiments, the pcDNA3.1 (empty expression vector, 1.5 µg/ml) was also transfected as a control for transfection effects. Following transfection, cells were maintained in serum-free DMEM. 48 h after transfection, cells were pulsed with 0.5 µCi/ml [methyl-3H]thymidine for 4 h before harvesting and assessing radioactivity. Panel A shows [ 3H]thymidine incorporation as a percent of control (nontransfected cells; NT). Values represent the mean ± S.E. (n = 15 separate transfections). Paired samples were compared by paired t test; *, values of p < 0.05 were considered significant. Panels B and C show representative Northern and Western blots of ATF3 mRNA and protein levels, respectively, following transfection (n = 3 independent experiments). The exogenous ATF3 mRNA produced by the pcDNA3.1/ATF3 plasmid (B, lanes 3-5) was smaller in size (~1 versus 2 kb) than the endogenous ATF3 mRNA expressed following serum stimulation (Panel B, lane 2). This expected size difference is a reflection of the fact that much of the 3'-untranslated region of the ATF3 gene was not included in the construction of the pcDNA3.1/ATF3 plasmid. In panels B and C, quiescent nontransfected cells were used as a negative control (Neg), and quiescent nontransfected cells stimulated with 10% FBS for 1 (B) or 3 h (C) were used as positive controls (Pos).

Northern (Fig. 3B) and Western (Fig. 3C) blot analysis demonstrated an increasing amount of ATF3 mRNA and protein, respectively, corresponding to increasing doses of pcDNA3.1/ATF3, whereas no ATF3 mRNA or protein was detected in the quiescent nontransfected control cells (lane 1) or in cells transfected with the maximum dose of the control plasmid (lane 6). It should be noted that the exogenous ATF3 mRNA produced by the pcDNA3.1/ATF3 plasmid (lanes 3-5) was smaller in size (~1 kb versus 2 kb) than the endogenous ATF3 mRNA expressed following serum stimulation (lane 2). This expected size difference is a reflection of the fact that much of the 3'-untranslated region of the ATF3 gene was not included in the construction of the pcDNA3.1/ATF3 plasmid, given that the parent vector already contained a bovine growth hormone polyadenylation signal (BGH pA, Fig. 2) to facilitate efficient transcriptional termination, polyadenylation, and enhanced stability of the exogenous ATF3 mRNA. The size difference between endogenous and exogenous ATF3 mRNA provided a useful means of confirming the efficacy of the expression vector and ensuring that the biological effects observed following transfection of this plasmid were indeed a result of controlled exogenous expression of ATF3.

Overexpression of ATF3 Leads to Increased DNA Synthesis in Response to Mitogenic Stimulation-- Once we had established that ATF3 was sufficient to stimulate DNA synthesis in Hepa 1-6 cells, we were interested in determining whether ATF3 overexpression would alter the serum responsiveness of these cells. Fig. 4A shows that Hepa 1-6 cells transfected with pcDNA3.1/ATF3 (1.5 µg/ml) consistently proliferated at a significantly higher rate than nontransfected cells or cells transfected with the control plasmid, both in the absence and presence of varying concentrations of FBS (1-20%). Northern (Fig. 4B) and Western (Fig. 4C) blot analysis confirmed the presence of exogenous ATF3 mRNA and protein, respectively, in the pcDNA3.1/ATF3 transfected cells but not in the nontransfected cells or in the cells transfected with the control plasmid.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Serum-dependent effects of exogenous ATF3 overexpression on [3H]thymidine incorporation (A), mRNA levels (B), and protein levels (C) in quiescent Hepa 1-6 cells. Cells were incubated in serum-free DMEM (36 h) before liposome-mediated transfection (12 h) with 1.5 µg/ml pcDNA3.1/ATF3 expression plasmid. Following transfection, cells were maintained in serum-free DMEM. 48 h after transfection, cells were cultured in the presence of FBS (0-20%) for 24 h and then pulsed with 0.5 µCi/ml [methyl-3H]thymidine for 4 h before harvesting and assessment of radioactivity. Panel A shows [ 3H]thymidine incorporation as a percent of control (nontransfected cells (NT)). Values represent the mean ± S.E. (n = 15 separate transfections). Paired samples were compared by paired t test; *, values of p < 0.05 were considered significant. Panels B and C show representative Northern and Western blots of ATF3 mRNA and protein levels, respectively, following transfection (n = 3 independent experiments). The exogenous ATF3 mRNA produced by the pcDNA3.1/ATF3 plasmid (B, lanes 3-5) was smaller in size (~1 kb versus 2 kb) than the endogenous ATF3 mRNA expressed following serum stimulation (B, lane 2). This expected size difference is a reflection of the fact that much of the 3'-untranslated region of the ATF3 gene was not included in the construction of the pcDNA3.1/ATF3 plasmid. In panels B and C, quiescent nontransfected cells were used as a negative control (Neg), and quiescent cells stimulated with 10% FBS for 1 (B) or 3 h (C) were used as a positive control (Pos).

Overexpression of ATF3 Enhances Endogenous Cyclin D1 mRNA Expression-- The present study has shown that overexpression of ATF3 stimulates DNA synthesis in Hepa 1-6 cells; however, the molecular mechanisms by which this occurs are unknown. To determine whether cyclin D1 was a molecular target of ATF3, we first examined the effect of ATF3 overexpression on cyclin D1 mRNA levels. Northern blot analysis (Fig. 5A) demonstrated increasing expression of endogenous cyclin D1 corresponding to increasing doses of pcDNA3.1/ATF3 (lanes 4-6), with low levels of cyclin D1 mRNA detected in the quiescent nontransfected control cells (lane 2) and in cells transfected with the maximum dose of the control plasmid (lane 3). Densitometric analysis confirmed that cyclin D1 expression was induced up to 2.3-fold in cells overexpressing ATF3, a level similar to that seen in nontransfected cells stimulated with 10% FBS (Pos., Fig. 5B).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Dose-dependent effects of exogenous ATF3 overexpression on cyclin D1 mRNA levels in quiescent Hepa 1-6 cells. Cells were incubated in serum-free DMEM (36 h) before liposome-mediated transfection (12 h) with 0.25, 0.5, or 1.5 µg/ml pcDNA3.1/ATF3 expression plasmid. 15 µg of total RNA was isolated for each time point and assessed by Northern blot analysis with a cDNA probe for cyclin D1. Load normalization was verified by reprobing blots with a 7 S RNA cDNA probe. Transfection of the pcDNA3.1 (empty expression vector, 1.5 µg/ml) was used as a control for transfection effects. Quiescent nontransfected cells stimulated with 10% FBS for 4 h were used as a positive control (Pos.). Panel A shows a representative Northern blot. Panel B shows the relative intensity of cyclin D1 mRNA expression following transfection with increasing amounts of pcDNA3.1/ATF3. The band intensities of three independent experiments were quantified densitometrically and expressed as a percentage of control (Neg, quiescence).

ATF3 Activates the Cyclin D1 Promoter by Binding to the AP-1 Consensus Site-- To determine whether the ATF3-dependent induction of cyclin D1 was being mediated at the transcriptional level, we co-transfected Hepa 1-6 cells with five different cyclin D1 reporter constructs (1.5 µg/ml; Fig. 6A) and the pcDNA3.1/ATF3 expression plasmid (100 ng/ml). Compared with control (cells transfected in the absence of pcDNA3.1/ATF3), overexpression of ATF3 increased reporter activity up to 2.6-fold in the 964CD1LUC and 1745CD1LUC wild-type promoter constructs, which both contained a region spanning intact AP-1 and CRE consensus sites from the cyclin D1 promoter (Fig. 6, A and B). In contrast, transfection of the 964AP-1mutCD1LUC and 1745CREmutCD1LUC promoter constructs containing a mutated AP-1 site (TGCGGCAG) or a mutated CRE site (TCGCGTCC) (38, 39) resulted in significantly lower induction of cyclin D1 promoter activity (1.3- and 1.6-fold, respectively) in the presence of pcDNA3.1/ATF3, although it was still greater than in control cells (Fig. 6, A and B) (bold italics denote mutated bases). Importantly, co-transfection with pcDNA3.1/ATF3 and the 1745AP-1/CREmutCD1LUC construct (in which both the AP-1 and CRE sites are mutated) resulted in complete abrogation of ATF3-mediated cyclin D1 promoter activation (Fig. 6, A and B). Co-transfection of the five cyclin D1 reporter constructs with a control plasmid (pcDNA3.1, empty expression vector) resulted in no change in promoter activity (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Activation of cyclin D1 by ATF3 in Hepa 1-6 cells. Panel A shows a schematic representation of the cyclin D1 promoter constructs in the vector pA3LUC, and indicates the areas homologous to the AP-1 (checkered oval) and CRE (gray oval) consensus elements, the transcriptional start site (black rectangle), and the luciferase coding sequence (striped box). Mutated promoter elements are indicated by an X. Quiescent cells were co-transfected (12 h) with 1.5 µg/ml of each of the cyclin D1 promoter constructs and 0 or 100 ng/ml of the pcDNA3.1/ATF3 expression vector. 48 h after transfection, cells were harvested and assayed for luciferase activity. Panel B shows the relative luciferase activity as compared with control (cell transfected in the absence of ATF3). Data are presented as the mean ± S.E. (n = 15 separate transfections). Paired samples were compared by paired t test; *, p < 0.01, and **, p < 0.05, represent a significant increase in promoter activity relative to control; , p < 0.01, represents a significant loss of promoter activity in the mutated constructs relative to their respective parental wild-type constructs.

To further characterize the role of ATF3 in cyclin D1 regulation, DNA binding studies were carried out to evaluate the interaction of ATF3 with the AP-1 and CRE consensus sequences contained within the cyclin D1 promoter. Electrophoretic mobility shift assays (Fig. 7A) demonstrated that nuclear proteins from serum-stimulated, nontransfected cells and ATF3-transfected cells formed specific complexes with both the CRE and AP-1 oligonucleotides (lanes 1, 2, 5, and 6). The specificity of these interactions was demonstrated by incubation with a 100-fold excess of unlabeled CRE or AP-1 oligonucleotides (lanes 3, 4, 7, and 8) and by incubation of these extracts with a nonspecific oligonucleotide, which did not produce a complex (data not shown).

View larger version (60K): [in this window] [in a new window]   Fig. 7.   ATF3 binds to the cyclin D1 AP-1 consensus sequence in Hepa 1-6 cells. A, electrophoretic mobility shift assays were carried out by incubating 25,000 cpm of -32P-labeled double-stranded oligonucleotides corresponding to the cyclin D1 CRE or AP-1 consensus sequences with nuclear extracts from cells transfected with 1.5 µg/ml pcDNA3.1/ATF3 (lanes 1-4) or nontransfected, serum-stimulated cells (lanes 5-8). The resulting complexes (lanes 1, 2, 5, and 6) could be blocked by a 100-fold excess of unlabeled CRE or AP-1 oligonucleotides (lanes 3, 4, 7, and 8). Incubation of extracts with an unrelated oligonucleotide did not produce a complex (data not shown). B, for immunodetection of ATF3, the binding reaction was carried out using 30-50 µg of nuclear extract from transfected (lanes 1-5) or nontransfected, serum-stimulated cells (lanes 6-10) and 0.6 µg/ml of biotinylated CRE and AP-1 probes identical to the probes used for the EMSA. The binding reactions were then incubated with streptavidin-agarose and washed, and the ATF3 protein bound to biotin-target DNA was collected in 1% SDS, 10 mM DTT and analyzed by Western blot analysis. The specificity of the DNA binding (lanes 2, 4, 7, and 9) was tested by addition of a 100-fold excess of nonbiotinylated CRE or AP-1 oligonucleotides (lanes 3, 5, 8, 10). Nuclear extracts incubated in the absence of oligonucleotide or streptavidin-agarose were used as positive controls (lanes 1 and 6). Panels A and B show representative gels (n = 3 independent experiments).

To determine whether ATF3 was the protein responsible for these DNA-protein complexes, a DNA binding assay based on the interaction of ATF3 with biotinylated cyclin D1 CRE and AP-1 consensus sequences was performed (Fig. 7B). This assay clearly and reproducibly demonstrated that ATF3 from both nontransfected and ATF3-transfected cells binds to the AP-1 site (lanes 4 and 9). No binding of ATF3 to the CRE site was detected in this assay (lanes 2 and 7). The specificity of the interaction between the cyclin D1 AP-1 site and ATF3 was demonstrated by competition with 100-fold excess of nonbiotinylated AP-1 (lane 5). To ensure that the biotinylated oligonucleotides interacted with the same proteins in the nuclear extracts, EMSA was performed as above and yielded complexes identical to those observed with the nonbiotinylated oligonucleotides (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The sequential, ordered expression of specific genes during the cell cycle suggests a stepwise genetic program for growth that is complex and highly regulated, and as such, may be prone to disruption in the case of injury or disease. Indeed, a major contributor to tumor development is the dysregulation of genes that control cellular growth processes and the resultant alteration to cell cycle control (34, 43). Because almost all of the cellular "decisions" regarding growth, differentiation, quiescence, and apoptosis occur during the G₁ phase of the cell cycle (43), it is crucially important to identify the molecular participants in the G₀/G₁ transition, the progression of G₁, and the G₁/S transition, as well as to elucidate the relationship that these molecules have to each other and to the various signal transduction pathways that together regulate the cell cycle.

The rapid and profound changes in hepatocellular growth during liver regeneration provide an important physiological model with which to study the molecular regulation of both normal and neoplastic cell growth. In this study, we present a useful in vitro model for the investigation of some aspects of hepatic cell growth. Hepa 1-6 mouse hepatoma cells demonstrate many of the morphological and biochemical markers characteristic of primary hepatocytes (36). We show here that the immediate-early gene ATF3 is expressed at very low levels in quiescent Hepa 1-6 cells but can be rapidly and highly induced by the addition of mitogens such as serum, HGF, or EGF. These results are entirely consistent with the findings of other studies, which demonstrate an almost identical expression pattern of ATF3 mRNA following dissociation and culture of primary hepatocytes (8),² partial hepatectomy (7, 10), or hepatotoxic insult with CCl₄ or alcohol (10). The consistency of ATF3 expression patterns among differentially induced hepatic growth situations implicates ATF3 as a key mediator of hepatocyte proliferation.

It has been proposed that ATF3 has an important role in regulation of the growth response in the regenerating liver by driving cells through the G₁ phase of the cell cycle via activation or repression of other growth-related genes (7). In the present study we defined, for the first time, a potentially significant biological role for ATF3 as a regulator of cellular proliferation. Overexpression of ATF3 leads to a dose-dependent increase in the DNA synthesis activity of both resting and mitogen-stimulated Hepa 1-6 cells, indicating that increased ATF3 can induce hepatocyte proliferation. It should be noted here that transfection efficiencies in this system are ~50%, suggesting that ATF3 may promote cellular proliferation to an even greater extent than reported here.

In addition to the effects on cellular proliferation, we have demonstrated that cyclin D1 mRNA expression is increased in cells that overexpress ATF3. It has previously been shown that cyclin D1 protein levels are strongly associated with changes in the steady-state levels of cyclin D1 mRNA (44), suggesting that the changes in mRNA levels observed here would also be reflected in increased cellular levels of cyclin D1 protein. Taken together with co-transfection experiments using an ATF3 expression vector and cyclin D1 promoter constructs, these data strongly suggest that the mechanism by which ATF3 drives hepatocyte proliferation is via transcriptional activation of the cyclin D1 promoter. The alteration of ATF3 binding by mutation of the AP-1 site (964AP-1mutCD1LUC construct) results in a reduction in activating ability, although ATF3 can still significantly enhance promoter activity compared with controls. Mutation of the CRE site (1745CREmutCD1LUC construct) also results in a significant reduction in activating ability, although to a lesser extent than demonstrated by mutation of the AP-1 site. Studies by Watanabe et al. (45, 46) support our findings that the cyclin D1 promoter can be activated primarily via the 953 AP-1 site with additional contribution from the 54 CRE site.

Electrophoretic mobility shift assays demonstrated that nuclear proteins from serum-stimulated, nontransfected cells and ATF3-transfected cells form specific complexes with both the AP-1 and CRE elements. However, immunodetection assays revealed a direct interaction between ATF3 and the AP-1 element only, with no ATF3 associating under these conditions with the CRE element within the cyclin D1 promoter. Combined with the reporter gene studies, these results may be interpreted in several ways. ATF3 may be acting indirectly on the cyclin D1 CRE site by binding to and activating another promoter target, of which the resultant protein then binds directly to the cyclin D1 CRE. Our observation that simultaneous mutation of both the AP-1 and the CRE sites contained within the cyclin D1 promoter leads to complete abrogation of ATF3-mediated promoter activation (1745AP-1/CREmutCD1LUC construct) indicates that there is an obvious requirement for an intact CRE site within the promoter. This suggests a potential functional and/or physical cooperation between the AP-1 and CRE promoter elements whereby ATF3 associates directly with the AP-1 element; a second, ATF3-activated factor is required at the CRE element, and both factors are required for enhanced cyclin D1 transcription. Alternatively, ATF3 may bind preferentially to the AP-1 site and to a lesser extent to the CRE site if both elements are present; and the assays that we used may not detect binding to the CRE site.

Taken together, the results presented here indicate that the greatest effect of ATF3 on cyclin D1 transcription involves the 953 AP-1 site. It has been demonstrated previously that this site is important in growth factor signaling (46, 47), suggesting that the cyclin D1 AP-1 site may be a critical participant in a number of molecular pathways involved with inhibition of apoptosis (47), cell cycle progression, and cellular transformation (45-47). In addition, a number of other studies have established that CREB (45, 48, 49) and Jun/Fos proteins (38, 45-47, 50) can transactivate the cyclin D1 promoter in proliferating cells by binding to the CRE and AP-1 sites, respectively, suggesting that these proteins may be involved directly or indirectly with the ATF3-dependent induction of the cyclin D1 promoter reported here. Studies are currently underway to further characterize the nature of the various nuclear factors that form complexes with both the AP-1 and the CRE sites within the cyclin D1 promoter in this cell system.

To date, only two other target promoters for ATF3 (gadd153/CHOP10 and ATF3 itself) (9, 16, 51) have been definitively identified. Although these studies point to important roles in the autoregulation of ATF3 activity, molecular targets for this factor that participate in the cell cycle or other aspects of cellular function have not yet been described. Our observation that ATF3 can activate the cyclin D1 promoter represents the first identification of an important cell cycle regulator that is responsive to ATF3. Cyclin D1 is a 35-kDa protein that is necessary for cell cycle progression, as demonstrated by experiments in which neutralization of cyclin D1 in early G₁ causes cellular growth arrest and an inability to progress through the remainder of the cell cycle (52, 53). Conversely, overexpression of cyclin D1 leads to acceleration of G₁ progression (54, 55). Although the mechanisms are not fully understood, the effects of cyclin D1 on cell cycle control probably relate to involvement in the p16-cyclin D1-retinoblastoma (Rb) pathway, a pathway that is often implicated in human neoplasia (24, 25). The Rb protein (pRb) has been referred to as the "gatekeeper" of the so-called R (restriction) point that occurs late in G₁ just prior to, but not concomitant with, the G₁/S phase transition (43). Once a cell has passed the R point, it is irreversibly committed to complete the remainder of the cell cycle (56). Hypophosphorylated Rb protein functions as an active growth suppressor in quiescent (G₀) cells but is progressively inactivated via phosphorylation by cyclin D1·CDK4/6 complexes as the cell progresses toward the R point. Constitutive hyperphosphorylation of Rb protein resulting from overexpression of cyclin D1 leads to loss of the R checkpoint, unscheduled entry into S-phase, and a tendency toward uncontrolled cellular proliferation (25). The elucidation of both upstream and downstream regulators of this pathway therefore appears highly important to the overall understanding of many cellular growth processes. We propose here that enhanced cyclin D1 expression in response to increased ATF3 levels drives cellular proliferation through modulation of this pathway. The precise nature of this modulation will require detailed investigation of the effects of ATF3 on cyclin D1 regulation and the effects of this transcription factor on other participants in cell cycle regulation. The presence of potential ATF3 binding sites in the promoter regions of other cyclins (57, 58) and of Rb itself (59, 60) suggests that several additional cell cycle-related genes may be subject to regulation by ATF3.

Our novel findings that ATF3 overexpression both activates the cyclin D1 promoter and confers upon quiescent cells the ability to enter S phase are highly consistent with the recognized roles of cyclin D1 in growth regulation. The observation that hepatic mitogens can induce endogenous ATF3 expression combined with the finding that ATF3 can enhance DNA synthesis makes ATF3 an attractive candidate as a molecular link in the transcriptional regulation of the cell cycle machinery in response to exogenous factors. For example, increased levels of cyclin D1 transcription appear to reduce cellular dependence on growth factors or other extracellular cues for proliferation (34), an observation consistent with our findings that Hepa 1-6 cells overexpressing ATF3 proliferate at a significantly higher rate both in the presence and absence of mitogenic stimuli. The roles played by other factors, including dimerization partners for ATF3 (AP-1 and CREB/ATF family proteins) and additional downstream targets that might be directly or indirectly regulated by ATF3, are also likely to be important in the complex network of cellular events that precede and accompany cellular proliferation.

	ACKNOWLEDGEMENTS

We thank Drs. Beth Drysdale and Allan Balmain for providing reagents used in this study. We thank James Gilmore for technical advice and Drs. Jacques Baudier and Spencer Greenwood for editorial comments and helpful discussion.

	FOOTNOTES

^* This work was supported in part by the Canadian Institutes of Health Research (CIHR) and by Grants R01CA70896, R01CA75503, and R01CA86072 from the Pfeiffer Foundation (to R. G. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ An Ontario Graduate Scholar.

A scholar of the Medical Research Council/CIHR. To whom correspondence should be addressed. Tel.: 519-824-4120, ext. 4935; Fax: 519-767-1450; E-mail: jlamarre@ovcnet.uoguelph.ca.

Published, JBC Papers in Press, May 24, 2001, DOI 10.1074/jbc.M103196200

² A. L. Allan and J. LaMarre, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: ATF, activating transcription factor; C/EBP, CCAAT/enhancer-binding protein; CREB, cyclic AMP response element-binding protein; CRE, cyclic AMP response element; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; EGF, epidermal growth factor; HGF, hepatocyte growth factor; PBS, phosphate-buffered saline; LUC, luciferase; Rb, retinoblastoma; bZIP, basic region and leucine zipper; AP-1, activating protein-1; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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