Glycogen synthase kinase 3β induces cell cycle arrest in a cyclin D1- dependent manner in human lung adenocarcinoma cell line A549

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1 204 Acta Physiologica Sinica, April 25, 2007, 59 (2): Research Paper Glycogen synthase kinase 3β induces cell cycle arrest in a cyclin D1- dependent manner in human lung adenocarcinoma cell line A549 LI Jian-Sha, ZHU Min, TIAN Dan, WANG Man-Xiang, WANG Fang, LI Na-Ping, WU Ren-Liang * Department of Pathology, Tongji Medical College, Huazhong University of Science and Technology, and Key Laboratory of Pulmonary Disease of Ministry of Health of China, Wuhan , China Abstract: The effect of glycogen synthase kinase 3β (GSK3β) has been repeatedly implicated in cell proliferation, but studies on the effect of GSK3β in different cell lines with different stimuli have drawn different conclusions. To investigate the direct effect of GSK3β on cell growth in human lung adenocarcinoma cell line A549, we changed its activity by transient transfection with two kinds of GSK3β mutant plasmids, constitutively active form S9A-GSK3β and dominant negative form KM-GSK3β. Twenty-four hours later, cell counting, flow cytometry and Western blot detection were made respectively. The results showed that enhancing GSK3β activity caused a decrease in cell number, as well as a higher percentage of cells at G 1 phase. Further, the expression of cyclin D1 was downregulated by GSK3β. Taken together, our observations suggest that GSK3β may induce G 1 cell cycle arrest in a cyclin D1-dependent fashion and therefore possibly plays a growth-inhibitory role in A549 cells. Key words: glycogen synthase kinase 3β; cell proliferation; cell cycle; cyclin D1 糖原合酶激酶 3β 以细胞周期蛋白 D1 依赖性方式引发人肺腺癌细胞 A549 细胞周期阻滞 * 李建莎, 朱敏, 田丹, 王满香, 王芳, 李娜萍, 吴人亮 华中科技大学同济医学院病理学系, 卫生部呼吸系统疾病重点实验室, 武汉 摘要 : 对糖原合酶激酶 3β (glycogen synthase kinase 3β, GSK3β) 在细胞增殖中的作用研究, 在不同细胞系和不同刺激因素作用下得出了不同结论, 本文旨在探讨 GSK3β 在人肺腺癌细胞系 A549 细胞生长中的直接作用 A549 细胞瞬时转染持续激活型 S9A-GSK3β 以及显性负突变型 KM-GSK3β 两种 GSK3β 突变型质粒, 改变 GSK3β 活性 24 h 后, 分别进行细胞计数, 流式细胞术及 Western blot 检测 结果显示, 增强 GSK3β 活性可导致细胞数量下降,G 1 期细胞百分比升高 细胞周期蛋白 D1 表达水平被 GSK3β 下调 结果提示,GSK3β 可能以细胞周期蛋白 D1 依赖性方式引发 A549 细胞的 G 1 期阻滞, 从而发挥生长抑制效应 关键词 : 糖原合酶激酶 3 β ; 细胞增殖 ; 细胞周期 ; 细胞周期蛋白 D1 中图分类号 :R322.3 Glycogen synthase kinase 3β (GSK3β) is a serine/threonine protein kinase that was first described in a metabolic pathway for glycogen synthase regulation [1]. In contrast to many other kinases, GSK3β is constitutively active in un-stimulated, resting cells and becomes inactive through phosphorylation at serine 9 by its upstream protein kinases. It has a variety of putative substrates, playing important roles in metabolism, cell proliferation, differentiation and survival [2]. GSK3β attracts more and more attention for its roles in a diverse range of cellular processes and its key position at several signaling pathways that are crucial in cancer and other human diseases. During embryonic hair follicle development, GSK3β may serve as a central signaling hub, allowing for tightly integrated and spatially con- Received Accepted This work was supported by the National Natural Science Foundation of China (No ). * Corresponding author. Tel: ; Fax: ; renliangwu@hotmail.com

2 LI Jian-Sha et al: GSK3β Induces G 1 Cell Cycle Arrest in A549 Cells 205 trolled proliferative responses [3]. Recent documents about tumors present opposing effects of GSK3β on cell proliferation. In a study of colon cancer cells, inhibition of GSK3β activity by chemical inhibitors and its expression by RNA interference targeting GSK3β induced apoptosis and attenuated proliferation, suggesting a possible role of GSK3β in promoting tumor cell survival and proliferation [4]. Conversely, in most investigations of tumors such as MCF- 7 breast cancer cells and hepatocellular carcinoma, GSK3β showed growth-inhibitory effect and therefore was predicted a tumor suppressor [5,6]. Lung cancer is the leading cause of cancer-related mortality in the world [7]. Its development is closely associated with dysregulation of Wnt signaling [8], in which GSK3β has been illustrated as a genuine switch that dictates both on and off states of a pivotal regulatory pathway [9]. However, little is known about the role of GSK3β in lung cancer. Our previous work has shown that GSK3β is rich in lung tissue and cultured pig bronchial epithelial cells [10]. Furthermore, we have demonstrated that this enzyme is involved in airway repair as well as squamous differentiation of airway epithelial cells [11-13]. In the present study, the direct effect of GSK3β on cell proliferation in the human lung adenocarcinoma cell line A549 was assessed. First we changed the activity of GSK3β by transient transfection with two GSK3β mutant plasmids, constitutively active form (S9A-GSK3β) [14] and dominant negative form (KM-GSK3β) [15]. Then we investigated the effects of changed GSK3β on cell growth as well as cell cycle progression in A549 cells. Our results supported a growthinhibitory role of GSK3β in A549 cells. 1 MATERIALS AND METHODS 1.1 Cell culture A549 cell line was obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). A549 cells were maintained in Dulbecco s modified Eagle s medium (DMEM) supplemented with 10% new calf serum (Zhejiang Sanli Biotechnology Company, China) and antibiotics (100 IU/mL penicillin and 100 µg/ml streptomycin) at 37 o C in 5% CO 2. Experiments were performed in the same passage of A549 cells and repeated at least 3 times. 1.2 Transient transfection The constitutively active GSK3β mutant (S9A-GSK3β) and dominant negative GSK3β mutant (KM-GSK3β) were generously provided by Professor James R. Woodgett (Ontario Cancer Institute, Canada). S9A-GSK3β was constructed by mutation of serine 9 to alanine using the palter-1 method [14], and the mutation renders the kinase insensitive to inhibitory phosphorylation and hence constitutively active. While KM-GSK3β, which was generated by altering the two consecutive lysine residues in the ATP-binding site to a methionine and an isoleucine, had no kinase activity and specifically interfered with endogenous GSK3β activity presumably by blocking access to substrates [15]. Therefore, it can be utilized as a dominant inhibitor of endogenous GSK3β. Transient transfection was carried out using Lipofectamine 2000 (Invitrogen, CA, USA) according to the recommendation from manufacturer and the method described by Tucker et al. [16] with minor modification. The cells were plated onto 6-well plate one day prior to transfection. Following confirmation of 70%-80% confluence, the cells were transfected with the GSK3β mutant plasmids (1 µg S9A-GSK3β and 1 µg KM-GSK3β, respectively) as well as empty vector control plasmid (1 µg pcdna3). Twenty-four hours later, a morphological observation was made, and then the cells were harvested for cell counting, flow cytometry, or the whole cell protein was extracted for a further Western blot detection. 1.3 Cell counting The cells were allowed to attach for 24 h at a density of about cells/well in 6-well culture plate. After transient transfection and a further culture for 24 h, the cells were washed with phosphate-buffered saline (PBS), incubated with 0.25% trypsin for 15 min at 37 o C. The digestion solution was mixed with 1.5 ml PBS, and aspirated repeatedly to make single cell suspension. Cell counting was performed using a hematocyte counting chamber. 1.4 Flow cytometry Twenty-four hours after transfection, the cells were washed with cold PBS, trypsinized down and collected by centrifugation, and then fixed with 80% cold ethanol at 20 o C overnight. Before measurement, the cells were washed, collected and re-suspended in PBS containing 10 µg/ml propidium iodide and 100 µg/ml DNase-free RNase A (Sigma, USA), and then incubated at 4 o C for at least 30 min. The percentage of cells at G 1, S and G 2 phases was determined by FACSCalibur (Becton Dickinson, USA). 1.5 Western blot analysis After the same duration of transfection, the cells were rinsed twice with cold PBS, collected by trpsinization and centrifugation, then lysed in buffer (50 mmol/l Tris-HCl, ph 8.0, 100 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l dithiothreitol, 1% Triton X-100, 0.1% SDS, 50 mmol/l sodium fluoride and 1 mmol/l sodium vanadate) containing a protease inhibitor cocktail to obtain the whole cell

3 206 protein. Protein concentration was determined by BCA kit (Pierce Chemical Company, USA). Equal amount of proteins were fractionated by SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membrane. The membranes were blocked with 5% nonfat milk in TBST and incubated with anti-gsk3β (1:1000, Santa Cruz, CA, USA), anti-phosphorylated GSK3β (1: 500, Cell Signaling, USA), anti-cyclin D1 (1:500, Santa Cruz, CA, USA) and anti-β-actin (1:1 000, Santa Cruz, CA, USA) antibodies overnight at 4 o C. The signal was detected by using a horseradish peroxidase-conjugated secondary antibody (Santa Cruz, CA, USA) and enhanced chemiluminescence (ECL, Pierce Chemical Company, USA), and then exposed to X-ray films (Fuji, Japan). 1.6 Statistical analysis Data were presented as mean±sd. One-way ANOVA test was used to determine significance for multiple group comparison (SPSS12.0 software). Differences were considered to be statistically significant at P< RESULTS 2.1 GSK3β inhibited proliferation of A549 cells To investigate whether GSK3β had a direct effect on A549 cell proliferation, we changed its activity by transient transfection with two kinds of GSK3β mutant plasmids for 24 h. Subsequently, we made a morphological observation of A549 cell growth. Under phase contrast microscope, the empty vector-transfected control cells looked spindle to round and stereo with close intercellular adhesion (Fig. 1A). Compared with that, the KM-GSK3β-transfected cells did not manifest distinct morphological changes except an increase in cell number (Fig.1C). Contrary to that, there was a great decrease in cell number in S9A-GSK3β group, and the cells became flattened, elongated and shrunken (Fig.1B). Additionally, a further cell counting signified S9A- GSK3 transfection might inhibit A549 cell proliferation while Acta Physiologica Sinica, April 25, 2007, 59 (2): KM-GSK3β transfection might promote cell growth (P< 0.05 compared with the control group) (Table 1). With Western blot analysis, it was confirmed that the Table 1. Effect of GSK3β on proliferation of A549 cells Control S9A-GSK3β KM-GSK3β Cell number ( 10 4 ) 39.3± ±3.4 * 50.8±8.7 * n=3 independent experiments done in duplicate. * P<0.05 as compared with the control group. different effects of GSK3β mutants on cell proliferation were caused by the change of kinase activity. Compared with that in the control group, GSK3β expression was distinctly enhanced in S9A-GSK3β group and slightly increased in KM-GSK3β group. Since GSK3β activation can be inhibited by serine 9 phosphorylation [2], we examined the inactive form of GSK3β with phosphorylation at serine 9 by Western blot with phosphospecific antibody. It showed a higher expression level of phosphorylated GSK3β in KM- GSK3β group but a significant decline in S9A-GSK3β group (Fig.2). The results were consistent with the functional characters of the GSK3β mutants, suggesting the plasmids we used were valid. Together with the data of cell counting, we deduced that GSK3β played an inhibitory role in A549 cell proliferation. 2.2 GSK3β induced G 1 cell cycle arrest in A549 cells In order to determine if the effect of GSK3β on A549 cell proliferation was due to interruption in cell cycle, cell cycle analysis was made using flow cytometry after the same duration of transfection. The result showed compared with that in the control group, the percentage of cells at G 1 phase increased significantly (P<0.01), whereas the population of cells at S phase decreased (P<0.05) in S9A-GSK3β group, indicating GSK3β activity may induce a G 1 cell cycle arrest in A549 cells. This possibility was further verified by the fact that, in KM-GSK3β group, there was a Fig. 1. Morphological and quantitative changes of A549 cells after transfection with GSK3β mutant plasmids under phase contrast microscope. A: Control group. B: S9A-GSK3β group. C: KM-GSK3β group. Scale bar, 50 µm.

4 LI Jian-Sha et al: GSK3β Induces G 1 Cell Cycle Arrest in A549 Cells 207 Fig. 2. Expression of GSK3β as well as phosphorylated GSK3β (p-gsk3β) in A549 cells after GSK3β mutant transfection. β- actin was used as a loading control. decreased population of cells at G 1 phase (P<0.05) while an increased population of cells at S phase (P<0.05) (Fig. 3). Based on these data, we conclude that GSK3 activity is critical for G 1 cell cycle arrest, and inhibition of the kinase activity might promote G 1 /S transition in A549 cells. 2.3 GSK3β caused cyclin D1 degradation Cyclin D1 is an important regulator of G 1 -S phase cell cycle transition and its proteolysis is thought to be mediated by GSK3β-induced phosphorylation and degradation [17]. Therefore, the total cell lysates were immunoblotted for Fig. 3. Effect of GSK3β activity change on cell cycle progression in A549 cells. A: Cell cycle analysis produced by flow cytometry. B: Percentages of cells at different phases in different groups. Data were obtained by densitometry measurement and presented as mean±sd. n=3. * P<0.05, ** P<0.01 vs the control group. Fig. 4. Expression of cyclin D1 in A549 cells after GSK3β mutant transfection. β-actin was used as a loading control. cyclin D1. The results showed cyclin D1 expression increased after KM-GSK3β transfection, but considerably decreased in S9A-GSK3β-transfected group (Fig.4). These data demonstrated that GSK3β might down-regulate cyclin D1 expression level by proteosomal degradation through phosphorylation.

5 208 3 DISSCUSSION GSK3β reportedly has opposing roles, inhibiting cell growth by repressing the Wnt/β-catenin signaling pathway on the one hand but maintaining cell survival and proliferation through the NF-κB pathway on the other hand. Previous studies on other cells with different stimuli have drawn different conclusions about the effect of GSK3β on cell proliferation [4-6]. Since this kinase may affect cell growth in a stimulus-dependent manner, in this study, we ruled out stimulating factors. GSK3β activity was directly changed by transient transfection with two GSK3β mutants, constitutively active form S9A-GSK3β and dominant negative form KM-GSK3β. Western blot analysis verified that the mutant plasmids were valid. GSK3β activity level was heightened in S9A-GSK3β group but lowered down in KM- GSK3β group. The following morphological observation and cell counting demonstrated a phenomenon that with increased GSK3β activity, the number of A549 cells decreased, suggesting a possible growth-inhibitory effect of GSK3β. Several studies reported that GSK3β mediates cell growth and proliferation via cell cycle regulation. During progressive hepatocarcinogenesis, increase in the level of inactive GSK3β contributed to the disruption of G 1 /S regulatory point in the cell cycle and thus leaded to abnormal cell proliferation [6]. In human breast cancer cell lines MCF-7 and MDA-MB-468, GSK3β was also critical for G 1 cell cycle arrest [5,18]. Our findings were consistent with the above studies. Elevating GSK3β activity by S9A-GSK3β transfection caused a larger population of A549 cells at G 1 phase and a reduction of cell percentage at S phase, implicating an essential role of GSK3β for G 1 cell cycle arrest; on the contrary, decreasing GSK3β activity with KM-GSK3β transfection promoted G 1 /S transition and therefore induced rapid cell growth. Cyclin D1 is an important regulator of G 1 -S phase cell cycle transition. It can bind to cdk4 and form a complex that phosphorylates several substrates, including Rb, and thus transcriptionally activates E2F target genes that trigger cells to progress from G 1 to S phase [19]. Meanwhile, cyclin D1 is also a known GSK3β substrate. GSK3β phosphorylates cyclin D1 at Thr-286, resulting in enhanced ubiquitylation, nuclear export and degradation of cyclin in the cytoplasm [17]. Previous work in several other cell lines implicated that GSK3β might inhibit cell proliferation by cyclin D1 down-regulation [5,6]. In this study, we showed an apparent increase of cyclin D1 expression in kinasedead KM-GSK3β-transfected A549 cells, but a significant Acta Physiologica Sinica, April 25, 2007, 59 (2): decline when GSK3β activity was increased by transfection with constitutively active S9A-GSK3β mutant, supporting such a possible mechanism that GSK3β might impact cell cycle progression through cyclin D1 degradation. However, a recent investigation on NIH3T3 cells indicated that changing GSK3β activity by LiCl, a selective inhibitor of GSK3β, had no effect upon cyclin D1 expression [20]. The causes of the discrepancy from our data might lie in different experimental methods, as well as different cell lines. 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