February 2007 Biol. Pharm. Bull. 30(2) 247—253 (2007) 247 Proteomics Analysis of the Proliferative Effect of Low-Dose Ouabain on Human Endothelial Cells a,b ,a a b c d Jie QIU, Hai-Qing GAO,* Rui-Hai ZHOU, Ying LIANG, Xu-Hua ZHANG, Xu-Ping WANG, a a Bei-An YOU, and Mei CHENG a Department of Geriatrics, Shandong University Qilu Hospital; 250012, Jinan, China: b Department of Intensive Care Unit, Shandong Qianfoshan Hospital; 250014, Jinan, China: c Experimental Center of Molecular Biology, Shandong University School of Medicine; 250012, Jinan, China: and d The Key Laboratory of Cardiovascular Remodeling and Function Research, Shandong University Qilu Hospital; 250012, Jinan, China. Received June 28, 2006; accepted November 15, 2006 Digitalis has been used to treat congestive heart failure for more than 200 years, although the dual effects (proliferation and death) induced by digitalis on cell growth have been known for many years, the mechanisms by which digitalis causes the actions were not completely known. The aim of this work was to characterize the proliferative effect of ouabain on cell growth in endothelial cells, and, to do the differential proteomic analysis of human umbilical vein endothelial cells (HUVEC) in response to ouabain and examine changes in protein expres- sion. HUVEC were exposed to different concentrations (0.1—100 nM) of ouabain at 12—48 h intervals. Cell growth and morphological changes of HUVEC treated with ouabain were compared with cells under nontreated conditions. Ouabain stimulated HUVEC cell proliferation at low concentrations and induced cell death at higher concentrations. Using proteomics study, we identified 32 proteins of HUVEC with various important cellular functions and revealed 8 proteins such as Annexin A1, Annexin A2, Malate dehydrogenase, Myosin regulatory light chain 2 (MRLC2), Profilin-1, S100 calcium-binding protein A13, Triosephosphate isomerase and Transla- tionally controlled tumor protein, regulated by low-dose ouabain treatment and MRLC2 was subsequently con- firmed by Western blot. Our results give new insights into the cellular and molecular mechanisms of the prolifer- ation action of low-dose ouabain on HUVEC and provide new avenues for the treatment of cardiovascular dis- eases. Key words ouabain; human umbilical vein endothelial cell; proteomics; proliferation; 2-DE; mass spectrometry Digitalis has played a prominent role in the therapy of con- In this study we focused on the effect of low doses of gestive heart failure since William Withering codified its use ouabain on proliferation in endothelial cells and did the dif- in 1785, the mechanisms of action of digitalis have been ferential proteomic analysis of human umbilical vein en- under extensive investigation for nearly 50 years, yielding dothelial cells (HUVEC) in response to low-dose ouabain. one of the most specific mechanisms thus far defined for any We compared the different protein expression of control cells agent so extensively used. Its ability to bind to and inhibit the and ouabain-treated cells and identified thirty-two proteins, Na-K-ATPase (NKA) has been well established, as has the in comparison with controls cells, the differential proteomic resulting increase in cellular [Ca2ϩ] responsible for its posi- analysis of HUVEC treated by ouabain further revealed the tive inotropic action and its toxicity as well. However, recent variation of eight proteins regulated by ouabain treatment observations suggest that digitalis has additional effects, such and expression of five of the protein spots increased and that as the positive inotropic effect on heart muscle, and the in- of three proteins decreased. This new information gives new ductive effect on cell growth, either hypertrophic on heart sights into the mechanisms for the proliferative effect of digi- muscle cells or proliferative on vascular smooth muscle talis on HUVEC and provides potential avenues for the de- cells.1) The inductive effect of cell proliferation by nontoxic velopment of future therapeutic interventions for the treat- concentrations of digitalis has been observed by Christen and ment of cardiovascular diseases. Dornand in studies on cell growth inducers for lymphocytes since 1975,2,3) and reports have published that ouabain exerts MATERIALS AND METHODS dual effects (proliferation and death) on cell growth at differ- ent concentration in different cell lines.4,5) Some researchers Materials Ouabain and 3-(4,5-dimethylthiazol-2-yl)- proposed the mechanism for ouabain’s proliferative inductive 2,5-diphenyltetrazolium bromide (MTT) were purchased effect is its interaction with NKA; this interaction occurred at from Sigma (U.S.A.), Human umbilical vein endothelial cells concentrations that did not induce enzyme activity inhibition (HUVEC) were obtained from American Type Culture Col- and that transformed it into a signal transducer system.4,6) lection (U.S.A.), cell culture medium was from Gibco However, experimental evidence has been put forward show- (U.S.A.), 2-DE reagents were obtained from Amersham Bio- ing that the effect of ouabain on cell survival and prolifera- sciences (U.S.A.) respectively unless otherwise indicated, tion may be either independent of or dependent on the inhibi- mouse monoclonal IgG antibody to MRLC2 was provided by tion of cation transport.7,8) All of these data indicate that Sigma-Aldrich, secondary antibody goat antimouse IgG was there maybe exist other target sites of digitalis in different purchased from Santa Cruz Biotechnology, and all the other cell lines besides NKA, and these target proteins play impor- chemicals used were of the highest grade available commer- tant roles in the regulation of cell growth and transformation cially. with or without interaction with NKA. Cell Culture The HUVEC were maintained in complete ∗ To whom correspondence should be addressed. e-mail: [email protected] © 2007 Pharmaceutical Society of Japan 248 Vol. 30, No. 2 medium M199 containing 10% FBS, 5 ng/ml bFGF, 25U/ml trace of bromophenol blue for the second treatment. Running heparin, 4 mML-glutamine, 100 U/ml penicillin-G, and conditions for horizontal SDS-PAGE were 20—50 mA for 100 U/ml streptomycin. Cells were cultured in Costar flasks approximately 2 h. and subconfluent cells were detached with 0.05% trypsin and Gel Staining and Spot Analysis Gels were stained with 0.02% EDTA in calcium-free phosphate buffered saline, silver according to Yan JX had reported.10) Briefly, the gels counted in hemocytometers and plated at 3ϫ103 in 100 ml were fixed in 50% ethanol and 5% acetic acid and then sensi- cultured in 96-well plate (for cell viability assay), or 75 cm2 tized in 0.02% sodium thiosulfate. The staining was per- Petri dish (for electron microscope and 2-DE) in complete formed in 0.1% silver nitrate. Dried 2-D gels were scanned M199 without bFGF. HUVEC were grown in complete using GS-800 Calibrated Densitometer (Bio-Rad) and image medium until reached about 80% of confluency and in most analysis as well as comparative studies were performed using cases such cells were used for the assays. Ouabain was dis- the PDQuest Image Analysis software (Bio-Rad). After solved in DMSO and diluted so that the final concentration background subtraction, normalization, and matching, the of DMSO was Ͻ0.1%, all the control cells were treated with spot volumes in gels from each control cells were compared same volume of DMSO, which caused no effect on cell with the matched spot volumes in gels from ouabain-treated growth. cells. Differential spots were selected when considering at Cell Viability Analysis To examine the effects of the least 100% of volume variation between matching spots in ouabain on HUVEC proliferation, cells were harvested by all three distinct comparative experiments. trypsinization and cell viability was measured by micro- In-Gel Trypsin Digestion and Analysis by Mass Spec- scopic examination of trypan blue dye exclusion. Cell sur- trometry Protein spots were in-gel digested by manually as vival was estimated with MTT colorimetric assay. HUVEC Shevchenko A has reported.11) Briefly, proteins were in-gel 6 (2ϫ10 cells/ml) were incubated in 24-well plastic culture reduced in 10 mM DTT and alkylated by 55 mM iodoac- plates for 12, 24 and 48 h at 37 °C in the presence or absence etamide, gel spots were washed by alternatively dehydrating of different concentrations of ouabain, MTT was added at gel pieces and finally dehydrated in acrylonitrile and dried a final concentration of 0.5 mg/ml. After being incubated for 15 min. Gel pieces were further rehydrated for 15 min at at 37 °C for an additional 4 h, cells were centrifuged at room temperature in a 0.5 mM solution of modified bovine 1000 rpm for 10 min and all the supernatants were discarded, trypsin and samples were incubated for 2 h at 37.7 °C. the cells were dissolved in 120 ml isobutanol (containing Adding 1% aqueous formic acid stopped the reaction; digests 0.04 mol/l HCl) and the absorbance was read at 570 nm, re- were desalted using Zip-Tips C18 (Millipore) and stepwise sults are presented as the average absorbance and expressed eluted in 50% and 100% acrylonitrile. The peptides mixture as the mean of six samples. was solubilized with 0.5% TFA for mass spectrometry analy- Morphology Examination To evaluate changes in mor- sis. The desalted peptide mixture volume was concentrated in phology, cells under the effect of ouabain were visualized by 2 ml of 1% aqueous formic acid. The extracted solutions transmission electron microscope (TEM). Logarithmic phase were mixed in an Eppendorf