糖原合酶激酶3β以cyclin D1依赖性方式引发人肺腺癌细

1、204Acta Physiologica Sinica, April 25, 2007, 59 (2): 204-209Research PaperGlycogen synthase kinase 3 induces cell cycle arrest in a cyclin D1-dependent manner in human lung adenocarcinoma cell line A549LI Jian-Sha, ZHU Min, TIAN Dan, WANG Man-Xiang, WANG Fang, LI Na-Ping, WU Ren-Liang*Department of

2、Pathology, Tongji Medical College, Huazhong University of Science and Technology, and Key Laboratory ofPulmonary Disease of Ministry of Health of China, Wuhan 430030, ChinaAbstract: The effect of glycogen synthase kinase 3 (GSK3) has been repeatedly implicated in cell proliferation, but studies on t

3、heeffect of GSK3 in different cell lines with different stimuli have drawn different conclusions. To investigate the direct effect of GSK3on cell growth in human lung adenocarcinoma cell line A549, we changed its activity by transient transfection with two kinds of GSK3mutant plasmids, constitutivel

4、y active form S9A-GSK3 and dominant negative form KM-GSK3. Twenty-four hours later, cellcounting, flow cytometry and Western blot detection were made respectively. The results showed that enhancing GSK3 activitycaused a decrease in cell number, as well as a higher percentage of cells at G1 phase. Fu

5、rther, the expression of cyclin D1 was down-regulated by GSK3. Taken together, our observations suggest that GSK3 may induce G1 cell cycle arrest in a cyclin D1-dependentfashion and therefore possibly plays a growth-inhibitory role in A549 cells.Key words: glycogen synthase kinase 3; cell proliferat

6、ion; cell cycle; cyclin D1糖原合酶激酶3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430030摘 要：对糖原合酶激酶3 (glycogen synthase kinase 3, GSK3)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GSK3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-GSK3以及显性负突变型KM-GSK3两种GSK3突变型质粒，改变

7、GSK3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GSK3活性可导致细胞数量下降，G1期细胞百分比升高。细胞周期蛋白D1表达水平被GSK3下调。结果提示，GSK3可能以细胞周期蛋白D1依赖性方式引发A549细胞的G1期阻滞，从而发挥生长抑制效应。关键词：糖原合酶激酶3；细胞增殖；细胞周期；细胞周期蛋白D1中图分类号：R322.3Glycogen synthase kinase 3 (GSK3) is a serine/threo-nine protein kinase that was first described in a metaboli

8、cpathway for glycogen synthase regulation1. In contrastto many other kinases, GSK3 is constitutively active inun-stimulated, resting cells and becomes inactive throughphosphorylation at serine 9 by its upstream protein kinases.It has a variety of putative substrates, playing importantroles in metabo

9、lism, cell proliferation, differentiation andsurvival2. GSK3 attracts more and more attention for itsroles in a diverse range of cellular processes and its keyposition at several signaling pathways that are crucial incancer and other human diseases. During embryonic hairfollicle development, GSK3 ma

10、y serve as a central sig-naling hub, allowing for tightly integrated and spatially con- Received 2006-11-23 Accepted 2006-12-29This work was supported by the National Natural Science Foundation of China (No. 30470757).*Corresponding author. Tel: +86-27-83692619; Fax: +86-27-83650729; E-mail: renlian

11、gwuLI Jian-Sha et al: GSK3 Induces G1 Cell Cycle Arrest in A549 Cells205trolled proliferative responses3. Recent documents abouttumors present opposing effects of GSK3 on cellproliferation. In a study of colon cancer cells, inhibition ofGSK3 activity by chemical inhibitors and its expressionby RNA i

12、nterference targeting GSK3 induced apoptosisand attenuated proliferation, suggesting a possible role ofGSK3 in promoting tumor cell survival and proliferation4.Conversely, in most investigations of tumors such as MCF-7 breast cancer cells and hepatocellular carcinoma, GSK3showed growth-inhibitory ef

13、fect and therefore was pre-dicted a tumor suppressor5,6.Lung cancer is the leading cause of cancer-related mor-tality in the world7. Its development is closely associatedwith dysregulation of Wnt signaling8, in which GSK3has been illustrated as a genuine switch that dictates bothon and off states of

14、 a pivotal regulatory pathway9.However, little is known about the role of GSK3 in lungcancer. Our previous work has shown that GSK3 is richin lung tissue and cultured pig bronchial epithelial cells10.Furthermore, we have demonstrated that this enzyme isinvolved in airway repair as well as squamous d

15、ifferentia-tion of airway epithelial cells11-13. In the present study, thedirect effect of GSK3 on cell proliferation in the humanlung adenocarcinoma cell line A549 was assessed. Firstwe changed the activity of GSK3 by transient transfec-tion with two GSK3 mutant plasmids, constitutively ac-tive for

16、m (S9A-GSK3)14 and dominant negative form(KM-GSK3)15. Then we investigated the effects ofchanged GSK3 on cell growth as well as cell cycle pro-gression in A549 cells. Our results supported a growth-inhibitory role of GSK3 in A549 cells.1 MATERIALS AND METHODS1.1 Cell cultureA549 cell line was obtain

17、ed from the Cell Bank of ChineseAcademy of Sciences (Shanghai, China). A549 cells weremaintained in Dulbeccos modified Eagles medium(DMEM) supplemented with 10% new calf serum (ZhejiangSanli Biotechnology Company, China) and antibiotics (100IU/mL penicillin and 100 µg/mL streptomycin) at 37 oC

18、in5% CO2. Experiments were performed in the same pas-sage of A549 cells and repeated at least 3 times.1.2 Transient transfectionThe constitutively active GSK3 mutant (S9A-GSK3) anddominant negative GSK3 mutant (KM-GSK3) weregenerously provided by Professor James R. Woodgett(Ontario Cancer Institute,

19、 Canada). S9A-GSK3 was con-structed by mutation of serine 9 to alanine using the pAlter-1method14, and the mutation renders the kinase insensitiveto inhibitory phosphorylation and hence constitutively active.While KM-GSK3, which was generated by altering thetwo consecutive lysine residues in the ATP

20、-binding site toa methionine and an isoleucine, had no kinase activity andspecifically interfered with endogenous GSK3 activitypresumably by blocking access to substrates15. Therefore,it can be utilized as a dominant inhibitor of endogenousGSK3. Transient transfection was carried out usingLipofectam

21、ine 2000 (Invitrogen, CA, USA) according tothe recommendation from manufacturer and the methoddescribed by Tucker et al.16 with minor modification. Thecells were plated onto 6-well plate one day prior totransfection. Following confirmation of 70%-80%confluence, the cells were transfected with the GS

22、K3mutant plasmids (1 µg S9A-GSK3 and 1 µg KM-GSK3,respectively) as well as empty vector control plasmid (1µg pcDNA3). Twenty-four hours later, a morphologicalobservation was made, and then the cells were harvestedfor cell counting, flow cytometry, or the whole cell pro-tein was extrac

23、ted for a further Western blot detection.1.3 Cell countingThe cells were allowed to attach for 24 h at a density ofabout 2×105 cells/well in 6-well culture plate. After tran-sient transfection and a further culture for 24 h, the cellswere washed with phosphate-buffered saline (PBS), incu-bated

24、with 0.25% trypsin for 15 min at 37 oC. The diges-tion solution was mixed with 1.5 mL PBS, and aspiratedrepeatedly to make single cell suspension. Cell countingwas performed using a hematocyte counting chamber.1.4 Flow cytometryTwenty-four hours after transfection, the cells werewashed with cold PBS

25、, trypsinized down and collected bycentrifugation, and then fixed with 80% cold ethanol at20 oC overnight. Before measurement, the cells werewashed, collected and re-suspended in PBS containing 10µg/mL propidium iodide and 100 µg/mL DNase-free RNaseA (Sigma, USA), and then incubated at 4 o

26、C for at least 30min. The percentage of cells at G1, S and G2 phases wasdetermined by FACSCalibur (Becton Dickinson, USA).1.5 Western blot analysisAfter the same duration of transfection, the cells were rinsedtwice with cold PBS, collected by trpsinization andcentrifugation, then lysed in buffer (50

27、 mmol/L Tris-HCl,pH 8.0, 100 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/Ldithiothreitol, 1% Triton X-100, 0.1% SDS, 50 mmol/Lsodium fluoride and 1 mmol/L sodium vanadate) contai-ning a protease inhibitor cocktail to obtain the whole cell206Acta Physiologica Sinica, April 25, 2007, 59 (2): 204-209protein. Pr

28、otein concentration was determined by BCA kit(Pierce Chemical Company, USA). Equal amount of pro-teins were fractionated by SDS-polyacrylamide gelelectrophoresis, and transferred onto nitrocellulosemembrane. The membranes were blocked with 5% non-fat milk in TBST and incubated with anti-GSK3 (1:1 00

29、0,Santa Cruz, CA, USA), anti-phosphorylated GSK3 (1:500, Cell Signaling, USA), anti-cyclin D1 (1:500, SantaCruz, CA, USA) and anti-actin (1:1 000, Santa Cruz,CA, USA) antibodies overnight at 4 oC. The signal wasdetected by using a horseradish peroxidase-conjugated se-condary antibody (Santa Cruz, CA

30、, USA) and enhancedchemiluminescence (ECL, Pierce Chemical Company,USA), and then exposed to X-ray films (Fuji, Japan).1.6 Statistical analysisData were presented as mean±SD. One-way ANOVA testwas used to determine significance for multiple group com-parison (SPSS12.0 software). Differences wer

31、e consi-dered to be statistically significant at P<0.05.KM-GSK3 transfection might promote cell growth (P<0.05 compared with the control group) (Table 1).With Western blot analysis, it was confirmed that theTable 1. Effect of GSK3 on proliferation of A549 cellsControl S9A-GSK3 KM-GSK3Cell numb

32、er (×104)39.3±5.221.6±3.4*50.8±8.7*n=3 independent experiments done in duplicate. *P<0.05 as com-pared with the control group.2 RESULTS2.1 GSK3 inhibited proliferation of A549 cellsTo investigate whether GSK3 had a direct effect on A549cell proliferation, we changed its activi

33、ty by transient trans-fection with two kinds of GSK3 mutant plasmids for 24h. Subsequently, we made a morphological observation ofA549 cell growth. Under phase contrast microscope, theempty vector-transfected control cells looked spindle toround and stereo with close intercellular adhesion (Fig.1A).

34、 Compared with that, the KM-GSK3-transfected cellsdid not manifest distinct morphological changes except anincrease in cell number (Fig.1C). Contrary to that, therewas a great decrease in cell number in S9A-GSK3 group,and the cells became flattened, elongated and shrunken(Fig.1B). Additionally, a fu

35、rther cell counting signified S9A-GSK3 transfection might inhibit A549 cell proliferation whiledifferent effects of GSK3 mutants on cell proliferationwere caused by the change of kinase activity. Comparedwith that in the control group, GSK3 expression wasdistinctly enhanced in S9A-GSK3 group and sli

36、ghtly in-creased in KM-GSK3 group. Since GSK3 activation canbe inhibited by serine 9 phosphorylation2, we examinedthe inactive form of GSK3 with phosphorylation at serine9 by Western blot with phosphospecific antibody. It showeda higher expression level of phosphorylated GSK3 in KM-GSK3 group but a

37、significant decline in S9A-GSK3group (Fig.2). The results were consistent with the func-tional characters of the GSK3 mutants, suggesting theplasmids we used were valid. Together with the data ofcell counting, we deduced that GSK3 played an inhibi-tory role in A549 cell proliferation.2.2 GSK3 induce

38、d G1 cell cycle arrest in A549 cellsIn order to determine if the effect of GSK3 on A549 cellproliferation was due to interruption in cell cycle, cell cycleanalysis was made using flow cytometry after the sameduration of transfection. The result showed compared withthat in the control group, the perc

39、entage of cells at G1phase increased significantly (P<0.01), whereas the popu-lation of cells at S phase decreased (P<0.05) in S9A-GSK3group, indicating GSK3 activity may induce a G1 cell cyclearrest in A549 cells. This possibility was further verifiedby the fact that, in KM-GSK3 group, there

40、was aFig. 1. Morphological and quantitative changes of A549 cells after transfection with GSK3 mutant plasmids under phase contrastmicroscope. A: Control group. B: S9A-GSK3 group. C: KM-GSK3group. Scale bar, 50 µm.LI Jian-Sha et al: GSK3 Induces G1 Cell Cycle Arrest in A549 Cells207decreased po

41、pulation of cells at G1 phase (P<0.05) whilean increased population of cells at S phase (P<0.05) (Fig.3). Based on these data, we conclude that GSK3 activity iscritical for G1 cell cycle arrest, and inhibition of the kinaseactivity might promote G1/S transition in A549 cells.2.3 GSK3 caused cy

42、clin D1 degradationCyclin D1 is an important regulator of G1-S phase cell cycletransition and its proteolysis is thought to be mediated byGSK3-induced phosphorylation and degradation17.Therefore, the total cell lysates were immunoblotted forFig. 2. Expression of GSK3 as well as phosphorylated GSK3(p

43、-GSK3) in A549 cells after GSK3 mutant transfection. -actin was used as a loading control.Fig. 3. Effect of GSK3 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 obtai

44、ned by densitometry measurement and presented asmean±SD. n=3. *P<0.05, *P<0.01 vs the control group.cyclin D1. The results showed cyclin D1 expression in-creased after KM-GSK3 transfection, but considerablydecreased in S9A-GSK3-transfected group (Fig.4). Thesedata demonstrated that GSK3 m

45、ight down-regulate cyclinFig. 4. Expression of cyclin D1 in A549 cells after GSK3 mutanttransfection. -actin was used as a loading control.D1 expression level by proteosomal degradation throughphosphorylation.2083 DISSCUSSIONGSK3 reportedly has opposing roles, inhibiting cell growthby repressing the

46、 Wnt/-catenin signaling pathway on theone hand but maintaining cell survival and proliferationthrough the NF-B pathway on the other hand. Previousstudies on other cells with different stimuli have drawndifferent conclusions about the effect of GSK3 on cellproliferation4-6. Since this kinase may affe

47、ct cell growthin a stimulus-dependent manner, in this study, we ruledout stimulating factors. GSK3 activity was directlychanged by transient transfection with two GSK3 mutants,constitutively active form S9A-GSK3 and dominant nega-tive form KM-GSK3. Western blot analysis verified thatthe mutant plasm

48、ids were valid. GSK3 activity level washeightened in S9A-GSK3 group but lowered down in KM-GSK3 group. The following morphological observationand cell counting demonstrated a phenomenon that withincreased GSK3 activity, the number of A549 cellsdecreased, suggesting a possible growth-inhibitory effec

49、tof GSK3.Several studies reported that GSK3 mediates cell growthand proliferation via cell cycle regulation. During progres-sive hepatocarcinogenesis, increase in the level of inactiveGSK3 contributed to the disruption of G1/S regulatorypoint in the cell cycle and thus leaded to abnormal cellprolife

50、ration6. In human breast cancer cell lines MCF-7and MDA-MB-468, GSK3 was also critical for G1 cellcycle arrest5,18. Our findings were consistent with the abovestudies. Elevating GSK3 activity by S9A-GSK3 trans-fection caused a larger population of A549 cells at G1 phaseand a reduction of cell percen

51、tage at S phase, implicatingan essential role of GSK3 for G1 cell cycle arrest; on thecontrary, decreasing GSK3 activity with KM-GSK3transfection promoted G1/S transition and therefore inducedrapid cell growth.Cyclin D1 is an important regulator of G1-S phase cellcycle transition. It can bind to cdk

52、4 and form a complexthat phosphorylates several substrates, including Rb, andthus transcriptionally activates E2F target genes that trig-ger cells to progress from G1 to S phase19. Meanwhile,cyclin D1 is also a known GSK3 substrate. GSK3 phos-phorylates cyclin D1 at Thr-286, resulting in enhancedubi

53、quitylation, nuclear export and degradation of cyclin inthe cytoplasm17. Previous work in several other cell linesimplicated that GSK3 might inhibit cell proliferation bycyclin D1 down-regulation5,6. In this study, we showedan apparent increase of cyclin D1 expression in kinase-dead KM-GSK3-transfec

54、ted A549 cells, but a significantActa Physiologica Sinica, April 25, 2007, 59 (2): 204-209decline when GSK3 activity was increased by transfec-tion with constitutively active S9A-GSK3 mutant, sup-porting such a possible mechanism that GSK3 might im-pact cell cycle progression through cyclin D1 degra

55、dation.However, a recent investigation on NIH3T3 cells indicatedthat changing GSK3 activity by LiCl, a selective inhibitorof GSK3, had no effect upon cyclin D1 expression20.The causes of the discrepancy from our data might lie in dif-ferent experimental methods, as well as different cell lines.Taken

56、 together, consistent with the results of studies inmost other cell lines, our findings for the first time demon-strated a possible growth-inhibitory role of GSK3 in A549cells by G1 cell cycle arrest, supporting that GSK3 might bea critical downstream effector of the antitumor effects18, 21.Therefor

57、e, potential approaches for the development ofpharmacological agents designed to increase GSK3 ac-tivity may lead to new strategies in lung cancer therapy.* * *ACKNOWLEDGEMENTS: The authors thank ProfessorJames R.Woodgett for the generous gifts of plasmids.REFERENCES1Cohen P, Nimmo HG, Proud CG. How

58、 does insulin stimulateglycogen synthesis? Biochem Soc Symp 1978; 43: 69-95.2Jope RS, Johnson GV. The glamour and gloom of glycogensynthase kinase-3. Trends Biochem Sci 2004; 29: 95-102.3Mill P, Mo R, Hu MC, Dagnino L, Rosenblum ND, Hui CC. Shhcontrols epithelial proliferation via independent pathways thatconverge on N-Myc. Dev Cell 2005; 9: 293-303.4Shakoori A, Ougolkov A, Yu ZW, Zhang B, Modarressi MH,Billadeau DD, Mai M, Takahashi Y, Minamoto T. DeregulatedGSK3beta activity