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 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 of HUVEC with various important cellular functions and revealed 8 proteins such as A1, , Malate dehydrogenase, Myosin regulatory light chain 2 (MRLC2), Profilin-1, S100 -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 [Ca2] 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 3103 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 0.1%, 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 (210 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 tube, and dried in a vacuum cells were trypsinized and plated to the subconfluent density concentrator. MS was performed on a liquid chromatography of 106/ml in 75 cm2 Petri dish and harvested after incubated electrospray ionisation mass spectrometry/mass spectrometry with different concentrations of ouabain. The cells were fixed (LTQ-ESI-MS/MS, ThermoFinnigan, San Jose, CA, U.S.A.), in 2.5% glutaraldehyde (v/v) and 2% osmium tetroxide using a surveyor high-performance liquid chromatography (w/v), washed in PBS, post-fixed in osmium tetroxide, dehy- (HPLC) system connected through PepFinder kit (with pep- drated in an ethanol series, embedded in epoxy resin, and ex- tide trap and 99 : 1 flow splitter), the ion transfer capillary amined under a Hitachi-700 TEM. temperature was set at 170 °C. Protein Sample Preparation Following stimulation was Database Searching and Functional Characterization achieved by incubation with ouabain 10 nM for 24 h in M199, of Identified Proteins Mass data collected during the 1107 cells were added to 100 ml lysis buffer (8 mol/l urea, LTQ-ESI-MS/MS analysis were processed and converted 4% CHAPS, 40 mmol/l Tris base). After a few cycles of into a file using the MasslynxTM software (Micromass) to be quick freezing and subsequent thawing in liquid nitrogen, submitted to the Mascot search software (http://www.matrix- 4 ml RNase (10 g/l) was added. After deposition for 20 min, science.com/). Identifications were obtained by comparison the cells were centrifuged at 12000 g for 30 min. The super- of experimental data with the NCBI non-redundant mam- natant was transferred to a new Eppendorf tube and stored. malian database and were validated when considering at least Protein concentrations were assessed using the protein assay two peptide sequences per protein. The main cellular location PlusOne 2-D Quant kit with BSA as the standard, then the and function of identified proteins, as well as their respective sample were divided into different EP centrifuge tube and biological relationships, were searched and determined by preserved at 80 °C. going through PubMed (http://www.ncbi.nlm.nih.gov/entrez/ Two-Dimensional Gel Electrophoresis Two-dimen- query.fcgi) with EndNote software v.5.0. sional electrophoresis (2-DE) and image analysis were per- Western Blotting Analysis of MRLC2 At the end of formed as previously reported with minor modifications.9) In stimulation, cells were washed, scraped from dishes and brief, isoelectric focusing (IEF) was carried out on an IPG- lysed in PBS. Proteins (30 mg) were separated by elec- phor using precast gel strips. After IEF, strips were equili- trophoresis in a 10% SDS-polyacrylamide gel. After the pro- brated twice for 10 min in equilibration buffer containing 6 M teins were transferred onto a polyvinylidene difluoride mem- urea, 30% v/v glycerol, 2% w/v SDS (Bio-Rad) in 50 mM brane (Millipore, Bedford, MA, U.S.A.), the blot was incu- Tris–HCl buffer, pH 6.8, supplemented with: (1) 60 mM DTT bated with blocking buffer (1PBS and 5% non-fat dry for the first treatment and (2) 259 mM iodoacetamide and a milk) for 1 h at room temperature and then probed with anti- February 2007 249

MRLC2 (1 : 200) monoclonal antibody, followed by incuba- well plates were treated with the indicated concentrations of tion with secondary antibody, horseradish peroxidase conju- ouabain at different intervals (Fig. 1A), MTT results showed gated affinity goat anti-mouse IgG (1 : 10000) for 1 h. To that the ouabain played a bifunctional role on growth of control equal loading of total protein in all lanes, blots were HUVEC, it increased HUVEC cell number when exposed to stained with mouse anti b-actin antibody at a 1 : 10000 dilu- concentrations 20 nM and induced cell at higher tion. The bound immunoproteins were detected by enhancer concentrations. Using the trypan blue exclusion assay, an in- chemiluminescent assay (ECL; Amersham), and the intensi- crease in cell number up to 40% of HUVEC treated with ties were quantified with a densitometric analysis (Digital 10 nM ouabain was observed when compared with the control Protein DNA Imagineware, Huntington Station, NY, U.S.A.). group (p0.01) as showed in Fig. 1B. Statistical Analysis Results are presented as means The Morphological Changes of HUVEC Exposed to standard error of the meansS.E. Statistical significance of Ouabain To support these results, we performed electronic the differences between experimental groups was calculated microscope analysis of HUVEC treated and not treated with by unpaired Student’s t-test, p0.05 was considered to be ouabain, the common features (Fig. 2A) and ouabain treated statistically significant. cells were observed under a transmission electronic micro- scope, cell proliferation were observed in 10 nM ouabain at RESULTS 24 h (Fig. 2B) while apoptosis were induced in 100 nM ouabain treated for 24 h (Fig. 2C). This result is accordance Effect of Ouabain on Cell Viability in HUVEC To test with results of the other experiments on the effect of low the effect of ouabain on HUVEC viability, the cells were doses of ouabain on different cell lines such as prostate cells, treated for 12, 24 and 48 h with various concentrations of Hela cells and vascular smooth muscle cells.4,5,8) ouabain (0.1, 0.5, 1, 5, 10, 20, 40, 100 nM) in a cell culture 2-DE and Mass Spectrometry To further investigate medium containing 10% (v/v) FBS. Cells growing on 96- the role of the inductive effect of low-dose ouabain on HUVEC proliferation, we pursued comparative proteomics analysis of endothelial cells in response to low-dose ouabain. Computer-assisted spot matching and silver staining images of the two gels, enabled localization of variation spots. Spots detected by silver staining were excised and subjected to for identification. In our experiments, we got the 2-DE of low- dose ouabain treated HUVEC (Fig. 3B) and control HUVEC (Fig. 3A); at least three 2-D gels were run per sample. Over 2800 protein spots visualized, 32 that were differentially ex- pressed in two groups were identified by LTQ-ESI-MS/MS, as shown in Fig. 4 and Table 1, the identified proteins in- volved in various aspect of endothelial cellular function, in- cluding metabolism, cell motility and as well. Among these proteins, in comparison with controls cells, the differential proteomic analysis of HUVEC treated by ouabain further revealed the variation of eight proteins, namely, , Annexin A2, Malate dehydrogenase, Myosin regulatory light chain 2, Profilin-1, S100 calcium- Fig. 1. Effect of Ouabain on Endothelial Cell Growth binding protein A13, Thioredoxin and Translationally con- (A) Cells (3103 in 100 ml) cultured in 96-well plates were exposed for 0, 12, 24 and trolled tumor protein (Table 2). Five proteins were over-ex- 48 h at indicated concentrations of ouabain. Cell proliferation was examined as de- pressed; they are Annexin A1, Annexin A2, Malate dehydro- scribed under Materials and Methods. Results are presented as the average absorbance and expressed as the mean of six samples. Differences between the groups were evalu- genase, Myosin regulatory light chain 2 and Profilin-1, while ated by Student’s t-test. (B) Cell viability were tested by examination of trypan blue dye three proteins were down-regulated, namely, S100 calcium- exclusion incubated with or without ouabain at 24 h. Results are expressed as relative cell number (%) with reference to control group. ∗ p0.01 compared with control binding protein A13, Thioredoxin, and Translationally con- group, ∗∗ p0.01 compared with each other group. trolled tumor protein.

Fig. 2. Picture of Transmission Electron Microscope (TEM) of HUVEC

(A) Control cells without ouabain. (B) Cells incubated with 10 nM ouabain at 24 h. (C) Apoptosis cells in the treatment with 100 nM ouabain for 24 h. White arrows showed nu- clei characterized by condensation and black arrows showed apoptotic bodies. M, mitochondria; N, nucleus; rER, rough endoplasmic reticula. 250 Vol. 30, No. 2

Fig. 3. Silver Stained 2-DE Picture (A) 2-DE profiles of total proteins extracted from the control cells. Comparison of both protein profiles revealed that there were many protein spots regulated. Over 2800 protein spots visualized 32 proteins that were identified by LTQ-ESI-MS/MS. (B) Picture of HUVEC with10nM ouabain-treated at 24 h. The number in gels is the spot identified.

Fig. 4. Representative Expanded Areas of Segments of 2D Gel Map (A—H) (A) Up regulation of annexin A1. (B) Up regulation of annexin A2. (C) Up regulation of malate dehydrogenase. (D) Up regulation of MRLC2. (E) Down regulation of thiore- doxin (absent from ouabain-treated cells). (F) Down regulation of S100A13 (absent from ouabain-treated cells). (G) Up regulation of profilin-1. (H) Down regulation of translation- ally controlled tumor protein.

Effect of Low-Dose Ouabain on the Expression of dose ouabain increased the expression of MRLC2 (p0.01), MRLC2 To conform the results of 2-DE and study the ex- meet the result of 2-DE as shown in Fig. 5. pression of MRLC2 in low-dose ouabain treated HUVEC, we carried out western blot analysis of MRLC2, and low- February 2007 251

Table1. Identified Proteins That Were Changed in Ouabain-Treated HUVEC

Numa) Accession number Protein name Mrb) Cover (%)c) PIb) Subcellular location

Cytoskeleton rearrangement 1 P62736 Actin 41736.77 29.07 5.29 Cytoplasm 2 O15511 Actin-related protein2/3 complex subunit 5 16189.19 40.00 5.47 Cytoplasm 10 Q02413 Desmoglein-1 precursor 113715.5 23.34 4.9 Membrane 21 P19105 Myosin regulatory light chain 2 19662.94 52.35 4.67 Cytoskeletal protein 23 P07737 Profilin-1 14922.95 31.65 8.47 Cytoskeletal protein 20 Q9Y2B0 MIR-interacting saposin-like protein 20652.29 35.93 4.81 Cytoskeletal protein 32 P02774 -binding protein precursor 52963.76 39.28 5.4 Secreted protein Regulation of cell metabolism 3 P00326 Alcohol dehydrogenase 39727.39 48.82 8.26 Cytoplasm 7 P25705 ATP synthase alpha chain 56559.96 38.83 5.26 Mitochondrion 19 P40926 Malate dehydrogenase 35531.25 40.71 8.92 Mitochondrion 11 P30084 Enoyl-CoA hydratase 31387.37 23.45 8.34 Mitochondrion 13 P07954 Fumarate hydratase 54636.87 21.20 8.85 Mitochondrion cytoplasm 28 P42765 3-Ketoacyl-CoA thiolase 41924.09 22.85 8.32 Mitochondrion 31 P60174 Triosephosphate isomerase 26712.44 67.87 6.9 Cytoplasm 17 P04196 Histidine-rich glycoprotein 59578.56 35.71 7.09 Secreted protein Calcium-binding protein 5 P04083 Annexin A1 38583.04 49.86 6.64 Cytosol 6 P07355 Annexin A2 38472.88 48.11 7.58 Secreted protein 8 Q9NZT1 -like protein 5 15920.58 21.23 4.34 Cytoplasm 16 P37235 Hippocalcin-like protein 1 22182.03 24.06 5.21 Cytoplasm 26 Q99584 S100 calcium-binding protein A13 11471.18 29.39 5.9 Cytoplasm; nucleus 30 P13693 Translationally controlled tumor protein 19595.35 26.86 4.84 Cytoplasm Nucleotide synthesis 9 P33316-1 Deoxyuridine 5-triphosphate nucleotidedohydrolase 26706.5 37.70 9.65 Nucleus; mitochondrion 12 Q9UBQ5 Eukaryotic translation initiation factor 3 subunit 12 25059.65 22.84 4.81 Nucleus 29 P22626 Heterogeneous nuclear ribonucleoproteins A2/B1 36005.98 19.45 6.22 Nucleus 25 P35244 Replication protein A 14 kDa subunit 13568.74 19.02 4.96 Nucleus Oxidative conversion 18 Q6W898 HSD33 46394.33 16.08 8.0 Cytosol 27 P10599 Thioredoxin 9451.96 38.82 5.77 Cytoplasm 14 P09382 Galectin-1 14584.5 35.37 5.34 Secreted protein 15 P10809 60 kDa heat shock protein 61212.59 46.78 5.7 Mitochondrion 22 Q99471 Prefoldin subunit 5 17328.08 21.43 5.94 Cytoplasm; nucleus Proteasome 4 P01023 Alpha2 macroglobulin 163237.9 11.33 16.06 Cytoplasm 24 P28072 Proteasome subunit beta type 6 25357.76 22.59 14.8 Cytoplasm; nucleus

a) Number of the protein spot in the 2-DE master gel. b) Theoretical Mr(Da)/pI are based on the amino acid sequence of the identified proteins and calculated with the Ex- PASy program. c) The sequence coverage of this protein with the matching peptides are indicated.

Table2. Summary of the Eight Proteins

Protein identification Mean vol. ratio: OU/Ca) S.D. Function

Annexin A1 3.14 0.19 Calcium/phospholipid-binding protein Annexin A2 2.58 0.20 Calcium-regulated membrane-binding protein Malate dehydrogenase 2.24 0.18 Catalyses the malate oxidated and produces NADH Myosin regulatory light chain 2 2.35 0.21 Plays an important role in regulation of cell contractile activity Profilin-1 2.19 0.16 Binds to actin and affects the structure of the cytoskeleton S100A13 Absent from OU Weakly binds calcium but binds zinc very tightly-distinct Thioredoxin Absent from OU Participates in various redox reactions Translationally controlled tumor protein 0.13 0.03 Involved in calcium binding and microtubules stabilization

a)Average volume ratio of the regulated proteins based on three independent experiments. OU: cells were treated with 10 nmol/l ouabain for 24 h; C: control cells.

DISCUSSION effect on cell proliferation with low concentrations of ouabain has been observed in studies mainly with ouabain in Ouabain, a digitalis compound, can regulate many cellular cell cultures of vascular smooth muscle of rat and dog, and events such as growth and proliferation. It modulates NKA recently in humans,5,12) as well as in human prostate smooth activity leading to the activation of different intracellular muscle cells.4) Furthermore, evidence was provided that pathways through protein–protein interactions that have been ouabain exerts dual effects (proliferation and apoptosis) on characterized during the last few years. Recent observations cell growth, a stimulating effect on cell proliferation has suggest that ouabain has additional effects on cardiac cell been observed at concentrations less than 10 nM, and ouabain function besides its positive inotropic action, the inductive at the concentrations are ineffective or only slightly affect the 252 Vol. 30, No. 2

related to cell mobility, signal transduction and proliferation, this new information also expand our understanding of the effect of ouabain on HUVEC at different concentrations. It is well known that endogenous digitalis-ouabain plays its role by inhibiting the a1 isoform of NKA, strikingly, one of the identified proteins expressed differently in ouabain- treated HUVEC, translationally controlled tumor protein (TCTP), also a NKA-binding protein serving as an intracel- lular inhibitor of NKA activity, acts on the cytoplasmic side of the .16) It has been shown to bind to tubulin in the cytoskeleton and is thought to be controlled at the translational as well as the transcriptional level.17) Further- more, we identified proteins implicated in signal transduction and membranes, with a large representation of multifunc- tional and S100A13, also support the idea that ouabain can develop its signal transduction functions without inhibit NKA. Annexin A1 plays a key role in mediating the Fig. 5. Effects of Low-Dose Ouabain on MRLC2 Expression (A, B) effects of and drugs on the immune and neuro-en- 18) HUVEC were incubated in the absence or in the presence of 10 nM ouabain for 24 h. docrine systems involved in signal transduction. Annexin (A) Cell lysates (20 mg) were processed for Western blotting analysis using a mono- clonal antibody to MRLC2 as described under Materials and Methods. (B) Relative A2 has been implicated in a number of biochemical amounts of MRLC2; Results were quantified by densitometry analysis of the bands and processes, including cell proliferation, ion-channel activa- then normalization to b-actin protein. Data are meansS.E. of four independent experi- tion, cell–cell interactions and the bridging of mem- ments, ∗ p0.01. branes.19,20) At the same time, Human S100A13, a ubiquitous protein involved in the cellular export of interleukin-1a (IL- Na /K exchange, while at concentrations above 10 nM, an 1a) and fibroblast growth factor-1 (FGF-1), plays a crucial inhibitory effect on cell growth is produced.4,5,7) Although role in angiogenesis and tissue regeneration.21) In addition, the proliferation induced by the NKA inhibitors such as the expression of thioredoxin and malate dehydrogenase, ouabain has been known for many years, the mechanism of which are related to nitrogen metabolism as components of this action was not well defined.7,8) Since the vascular en- the tricarboxylic acid cycle were also regulated, and this dothelium could be a potential target of endogenous ouabain, might also induce NO production. Thioredoxin, involved in we investigated the effect of ouabain on proliferation in the first unique step in DNA synthesis, also provides control HUVEC. over a number of transcription factors affecting cell prolifera- In cell viability assay, we observed that ouabain stimulated tion and death through a mechanism referred to as redox reg- cell proliferation and increased cell viability at low concen- ulation,22) more interesting, among the finally thioredoxins trations, also the stimulation of cell proliferation at concen- activate numerous target enzymes by reduction of disulfides, trations below 10 nM and induction of apoptosis at higher one of these target enzymes is the NADP-dependent malate concentrations were confirmed by TEM, similar biphasic be- dehydrogenase.23) Thus, it is conceivable that alterations in havior has been shown in the ouabain-induced mitogenic ef- these proteins might stimulate the cell growth and metabo- fect on VSMC12) as well as in human prostate smooth muscle lism of HUVEC. cells.13) To further investigate the effect of low-dose ouabain Meanwhile, we got the variation of profilin-1 and myosin on endothelial cell proliferation, we compared different pro- regulatory light chain 2 (MRLC2), though profilin-1 is tein expression of the ouabain-treated HUVEC with control thought to play a key role in the regulation of actin group. Despite the fact that 2-DE probably did not allow the dynamics,24,25) there is growing evidence that, apart from efficient separation of the majority of hydrophobic proteins actin binding, it acts as hubs that control a complex network such as membrane receptors or proteins from the mitochon- of molecular interactions involved in membrane trafficking, dria,14) the functional characterization of the identified pro- small-GTPase signaling and nuclear activities, and plays a teins illustrate some morphological and biological features of pivotal role at the interface of the phosphoinositide signal the effect of ouabain, such as cell contractility, accommodate transduction pathway and the cytoskeleton.26) MRLC2,27,28) of Ca2, cellular metabolism, signal transduction, resistance which was found to be up regulated in the present study, has to chemical or physical “stress,” and antigen processing by important effect on cell contraction and signaling pathway. proteasome (Fig. 4, Tables 1, 2). Ren reported the results of Recent research paid more interest in the role of myosin reg- mRNA profile analysis of HUVEC with ouabain treatment at ulatory light chain 2 in actin reorganization.29,30) Therefore, pathological concentrations (0.9—1.8 nmol/l),15) the regu- in the present paper, we only focused on MRLC2, in our ex- lated were not accordance with the proteins identified periment, we found that MRLC2 was up-regulated in the in this experiment, we think the discrepancy may be due to treatment of low doses of ouabain and the western blot assay cell type, the concentration of ouabain and the techniques obtained results similar to 2-DE, the expression of MRLC2 used. On the other hand, the regulated genes identified using increased in the low doses of ouabain-treated HUVEC com- mRNA profiling were related to signal transduction and me- pared with control group, and the unregulated MRLC2 tabolism, the differential proteomic analysis of HUVEC played an important role in the HUVEC proliferation without treated by low-dose ouabain further revealed the variation of the inhibition of NKA. Further work is underway. eight proteins involved in the various cellular functions more Taken together, we found digitalis played its role in the February 2007 253 cell proliferation, cell motility, signal transduction and other Proteomics, 3, 64—72 (2003). cellular processes in endothelia cells by different means, the 10) Yan J. X., Robin W., Tom B., Electrophoresis, 21, 3666—3672 (2000). identified proteins offer possible sites for therapeutic inter- 11) Shevchenko A., Wilm M., Vorm O., Mann M., Anal. 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