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1)Department of Orthopedic Surgery, Nagoya City University Medical School
2)Department of Bioregulation Research, Nagoya City University Medical School
3)Nagoya City University School of Nursing
Address for Correspondence: Yoshihiro Shibata, Department of Orthopedic Surgery, Nagoya City University Medical School, 1 Kawasumi, Mizuho-ku , Nagoya 467-8601, Japan. Phone 052-853-8236, Fax: 052-842-0266
Running title: Expression of ATBF1 in the lung
Key Words: ATBF1, homeodomain, zinc finger, A549, lung

The transcription factor ATBF1, a multiple-homeodomain zinc finger protein, is highly expressed in various tissues including the developing sensory nervous system, the small intestine and embryonic and adult lung. A histological analysis should give us insight into the function of ATBF1 in the lung. We investigated the localization of ATBF1 in adult mouse lung by in situ hybridization using cRNA and found expression of ATBF1 mRNA in the layer of epithelial cells of bronchioli and bronchi. The A549 cell line, which was derived from a type II pneumocyte tumor, was employed to study the patterns of expression of ATBF1 mRNA under various culture conditions. Although the expression levels of ATBF1 mRNA did not alter during progression of the cell cycle, there was a tendency for A549 cells transfected with ATBF1 cDNA expression vector to accumulate at the G0/G1phase

The ATBF1(AT motif binding factor 1) gene was first isolated from human hepatoma cells based on the ability of its product to bind to an AT-rich enhancer element of the human a-fetoprotein gene (1). The encoded protein, now referred to as ATBF1-B, is characterized by a large molecular weight (306 kDa) and the presence of four homeodomains (I-IV) and 18 zinc fingers including one pseudo zinc finger motif. More recently, we isolated a second ATBF1 cDNA termed ATBF1-A from human embryonic lung fibroblasts (2). This cDNA differs from ATBF1-B cDNA by a 3.3-kb extension at its 5' end which encodes five additional zinc finger motifs. We also cloned a murine homologue of the human ATBF1-A cDNA (3). High levels of ATBF1 mRNA were found in mouse embryo brain and in the lung(4). Although the level of ATBF1 mRNA decreased in the adult nervous system, it was maintained in the adult lung. There was a report of embryonic death of ATBF1 deficient mice caused by malfunction of the lung at birth (Noguchi et al., 20th annual meeting of the Molecular Biology Society of Japan, unpublished data). Although the knockout (ATBF1(-/-)) embryos grew to the final stage of gestation period, they died without respiratory activity at birth. The manifestation of the fetal death was similar to the neonatal respiratory distress syndrome that is related to infantile prematurity of the lung. We speculated that there are important roles for ATBF1 in the maturation of the lung, especially type II pneumocytes. One of the important factors for the initial expansion of pulmonary alveoli is the production of surfactant composed principally of phosphatidylcholine secreted by type II pneumocytes. We employed the A549 cell line that was derived from a type II pneumocyte tumor (5) as a model of pulmonary differentiation. The cell line is expresses type II properties, such as production of pulmonary surfactant (6) and can be induced to differentiate by a paracrine factor, or factors, released from human fetal lung fibroblasts under the control of dexamethasone (7). In this study, we investigated the pattern of expression of ATBF1 in adult mouse lung and analyzed ATBF1 mRNA levels in A549 cells cultured under various conditions.

In situ hybridization
Digoxigenin-labeled cRNA probes (2) for mouse ATBF1 were prepared from mouse ATBF1 cDNA cloned into pBluescriptIIKS(+) (Stratagene, CA, USA) using a DIG RNA Labeling kit (Roche, Basel, Switzerland). In situ hybridization was performed using the anti-sense strand and the sense strand as negative controls with 4% paraformaldehyde-fixed and paraffin-enbedded tissue sections of adult C57BL/6 mouse lung.
Cell culture
A549 human type II pneumocyte tumor cells were obtained from the Japan Health Sciences Foundation and were grown in Eagle's minimal essential medium (MEM) containing 10% fetal bovine serum. Culture with 0.5% fetal bovine serum for 48 hr caused cell cycle arrest. Synchronization of A549 cells was achieved by blocking metabolism by treatment with 100mg/ml hydroxyurea (Sigma-Aldrich, MO, USA) for 24 hr. The time when hydroxyurea was washed from the medium was considered as time 0.
RNA extraction and RT-PCR
Total RNA was prepared by a method based on
the guanidine thiocyanate procedure using TRIzolTM Reagent (GIBCO BRL, MD, USA). The first strand cDNA was synthesized using reverse transcriptase, SUPERSCRIPT II (GIBCO BRL, MD, USA) with random hexamers. PCR amplification was performed using primers for human ATBF1 (5' primer, TTGGAAGAGGCAGGAAAGCAG; 3' primer, GATTGGAGGTGGTAAAGGTGTT), for human c-Myb (5' primer, TCTTCTGCTCACACCACTGG ; 3' primer, GGCTGAGAATGCATTCACG), and for human glyceroaldehyde 3-phosphate dehydrogenase (GAPDH) (5' primer, CGGATGCAACGGATTTGGTCGTAT; 3' primer, AGCCTTCTCCATGGTGGTGAAGAC). The primer sets were designed using the analysis program Primer 3 from the internet web site
Transfection and Cell cycle analysis by FACScan
A549 cells were harvested and resuspended in Krishan's solution (propidium iodide (PI) 50mg/ml, sodium citrate 0.1%, ribonuclease A 20 mg/ml, Igepal CA-630 (Sigma-Aldrich, MO, USA) 0.3%) and analyzed directly by FACScan (Becton Dickinson, NJ, USA). We prepared forward and reverse oriented ATBF1 cDNA expression vectors, pATBF1-A and pATBF1-Rev respectively. The cells were transfected with purified plasmid DNA as calcium phosphate-DNA precipitate in N, N-bis-(2-hydroxyethyl)-aminoethanesulfonic acid (BES) buffer and incubated for 16 hr in an atmosphere containing 3% CO2 (8). The cells were also cotransfected with a green fluorescent protein (GFP) expression vector pEGFP-C1 (Clontech, CA, USA) as a marker of transfection. Fluorescein isothiocyanate (FITC)/PI double staining was performed after the cells were fixed with ethanol. The cells were incubated for 1hr at room temperature with anti-GFP rabbit IgG (Molecular Probe, OR, USA) in phosphate buffered saline containing 0.1% Tween 20(TPBS) containing 1% bovine serum albumin and 500 mg/ml of RNase A. After washing with TPBS, the cells were incubated for 1hr at room temperature with FITC conjugated anti-rabbit IgG(H+L)-goat antibody (ICN, CA, U.S.A.) and 500 mg/ml of RNase A, washed with TPBS three times and then stained with 400 mg/ml of propidium iodide solution (Sigma-Aldrich, MO, USA). The GFP positive cells after transient transfection with pATBF1-Rev and pEGFP-C1 were selected as a control group and were 1.5 % of the total number of 460450 cells analyzed by FACScan (Fig. 4a). The GFP positive cells after transient transfection with pATBF1-A and pEGFP-C1 were analyzed as ATBF1 expressing cells and were 2.2 % of the total number of 376885 cells (Fig. 4b).

ATBF1 mRNA was detected in the layer of epithelial cells of the bronchioli of adult mouse lung (Fig.1b).
A549 cells, a human lung adenocarcinoma cell line, were synchronized to determine ATBF1 expression during specific periods of the cell cycle. A549 cells were synchronized effectively by imposing a metabolic block with hydroxyurea (Fig. 2a). The expression of c-myb mRNA increased when the cells were released from the metabolic block (Fig. 2c). In contrast to c-myb mRNA, ATBF1 mRNA was not altered significantly though progression of the cell cycles (Fig. 2b). To confirm the expression of ATBF1 mRNA levels during the cell cycle, we blocked progression of the cell cycle by serum deprivation. A549 cells were cultured for 48hr in Eagle's MEM containing 0.5% serum to accumulate the cells at the G1 phase of the cell cycle (Fig. 3a). The population of the cells in the S phase was increased by 10% serum in the medium (Fig. 3b). We could not find a relationship between the level of ATBF1 mRNA and the concentration of serum in the medium (Fig. 3c). Although the data were not significant (p=0.11), c-myb mRNA showed a tendency to decrease in medium containing 0.5% serum (Fig. 3d).
We observed that 67.2% and 77.3% of the cells were accumulated in the G0/G1 phase when they were transfected with mock vector or with ATBF1 cDNA respectively. The coefficient of variations (CV) of the mean value, which is an indicator of the uniformity of the cells investigated, was 12.2 for the control (Fig. 4a) and 11.9 for the ATBF1 positive group (Fig. 4b). Although there was a tendency for A549 cells transfected with ATBF1 cDNA expression vector to accumulate in G0/G1 phase, we could not conclude that there was a significant influence ATBF1 because of the large CV values (Fig. 4a, 4b).

The level of ATBF1-A mRNA highly elevated in early development (9) as well as in adult lung (3). To characterize the ATBF1 positive cells, it is important to investigate the localization of ATBF1 mRNA in the lung. The localization of ATBF1 mRNA was determined by in situ hybridization to be in the layer of epithelial cells of bronchioli. Although several type II pneumocytes in normal adult alveoli should contain ATBF1 mRNA for the production of pulmonary surfactant, we only detected a few ATBF1 mRNA positive cells in the alveoli. This is not consistent with our initial idea that mature type II pneumocytes would highly express ATBF1 for the production of pulmonary surfactant.
We employed the A549 cell line derived from a type II pneumocyte tumor to investigate the mechanisms of pulmonary differentiation. Type II pneumocytes are not only important for the production of pulmonary surfactant but also for regenerative activity to replace destroyed pneumocytes (10). We used A549 to investigate the roles of the ATBF1 during progression of the cell cycle. We demonstrated that ATBF1 was not altered in the various phases of the cell cycle. We only observed a subtle tendency of inhibition of progression of the cell cycle by transient transfection with ATBF1 cDNA in A549 cells. We may have analyzed cells with and without ATBF1 cDNA expression vectors because of the indirect selection using GFP protein as a marker.
Recently, we suggested that the absence of ATBF1 might be responsible for the highly malignant nature of a-fetoprotein producing gastric cancers (11). The mechanism for this may be that ATBF1 represses the transcriptional activity of c-Myb by protein-protein interaction (12). The oncoprotein, c-Myb, is associated with growth and survival of cells through induction of bcl-2 (13). The absence of ATBF1 should help cells survive by the induction of bcl-2. Conversely, elevated ATBF1 may result in susceptibility to apoptosis because of the suppression of bcl-2 transcription. We observed a dramatic elevation of ATBF1 mRNA in P19 mouse embryonal carcinoma cells during induction of neuronal differentiation with retinoic acid (2, 14). Among the various molecules affecting apoptosis, expression of bcl-2 was selectively suppressed during retinoic acid-induced apoptosis of P19 cells (15). Thus, ATBF1 could function as one of the triggers of apoptosis.
ATBF1 should participate in the developmental differentiation of lung because of the high level of ATBF1 mRNA in embryonal mouse lung probably due to its apoptosis-inducing effect. The absence or very low levels of ATBF1 mRNA expression in alveolar epithelia of adult mouse resembles the suppression of ATBF1 mRNA in the adult CNS and fully differentiated mucosal upper epithelia of the brush-border of the small intestine (16). ATBF1 positive cells are plentiful in the CNS at early developmental stages, which may relate to the potential susceptibility to apoptosis. It is also highly expressed in the epithelia of the lower part of the crypt of the small intestine in the adult mouse (16). Here, we only determined the localization of cell expression ATBF1 mRNA in the lung, but we do not know the target of ATBF1 during lung differentiation. Further investigation is needed to determine the meaning of the high expression of ATBF1 mRNA in the lung.

We thank M. Yamamoto for excellent technical assistance. This work was supported by a research grant from the Aichi Prefecture Cancer Research Promoting Foundation.

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Fig. 1
Identification of cells expressing ATBF1 mRNA cells in adult mouse lung. In situ hybridization using the sense strand of ATBF1 cRNA (a) as negative control. The expression of ATBF1 mRNA (b) in the layer of epithelial cells of bronchioles using anti-sense ATBF1 cRNA.

Fig. 2
Analysis of ATBF1 and c-myb mRNAs transcripts in various phases of the cell cycle. a, Propidium iodide (PI) staining of A549 cells. The cells were synchronized by treatment with hydroxyurea (100mg/ml) for 24 hr, and then washing them with normal growth medium. Time indicates the time after washing with normal growth medium. Analysis of ATBF1(b) and c-myb (c) mRNAs by RT-PCR. PCR products were electrophoresed on agarose gels, and then stained with ethidium bromide. The intensities of the bands were measured with NIH image. The circles indicates the mean values. Standard errors from three independent measurements are indicated by bars. There was no significant alteration (p>0.05) in ATBF1 gene expression during progression of the cell cycle, but c-myb mRNA was elevated in cells in MEM containing 10% serum (p<0.05)

Fig. 3
Expression levels of ATBF1 and c-myb mRNA during serum deprivation and in normal growth medium. Propidium iodide (PI) staining of A549 cells grown in MEM containing 0.5% (a) or 10% (b) serum. Analysis of ATBF1 (c) and c-myb (d) mRNAs by RT-PCR. PCR products were electrophoresed on agarose gels and then stained with ethidium bromide. The intensity of the bands were measured with NIH image. The standard errors from three independent measurements are indicated by bars at the mean values.

Fig. 4.
FACScan analysis of A549 cells transfected with ATBF1 cDNA expression plasmids and reverse oriented ATBF1 cDNA expression plasmids as a negative control. a, With control cells 67.2% were accumulated in the G0/G1 phase. b, With cells transiently transfected with of ATBF1 cDNA 77.3% were accumulated in the G0/G1 phase.

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