Comparative Proteomic Analysis of Proteins Involved in Cell

Comparative Proteomic Analysis of Proteins Involved in Cell Aggregation during Neural Differentiation of P19 Mouse Embryonic Carcinoma Cells Xia Gao,† Hong-Yu Tian,† Li Liu,‡ Mei-Lan Yu,# Nai-He Jing,*,‡ and Fu-Kun Zhao*,†,# State Key of Molecular Biology and Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China, and College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China Received October 21, 2008

Cell-cell interactions play a crucial role during embryogenesis and are enhanced during cell aggregation. P19 mouse embryonic carcinoma cells can differentiate into neural cells by the addition of retinoic acid (RA) or by overexpression of the Wnt1 gene, with both processes dependent on cell aggregation. To identify molecules involved in the cell aggregation process, two-dimensional gel electrophoresis (2DE) was used to establish the cell aggregation-associated protein profiles. MALDI-TOF/TOF was used to identify 71 protein spots with differential expression patterns. Among these spots, 54 were differentially expressed in both P19 and Wnt1-overexpressing P19 (Wnt1/P19) cell aggregates, with 42 proteins up-regulated and 12 proteins down-regulated. The other 17 spots were differentially expressed only in Wnt1/P19 cells. The expression patterns of 5 cell aggregation-associated proteins, N-myc downstream-regulated gene 1 (NDRG1), 14-3-3 epsilon, 14-3-3 gamma, acid calponin and cell division control protein 2 homologue (Cdc2), were confirmed by immunoblot and RT-PCR. To further investigate the relationship between cell aggregation and neural differentiation, NDRG1 expression was inhibited by RNA interference during P19 cell aggregation. Silencing of NDRG1 reduced the size of cell aggregates and the expression of N-cadherin, and it also impaired the RA-induced P19 cell neural differentiation. In conclusion, this study provides new clues for the possible mechanism underlying cell aggregation during pluripotent stem cell neural differentiation. Keywords: P19 cell • Cell aggregation • Neural differentiation • Comparative proteomics • NDRG1 • RNA interference

Introduction Three-dimensional interactions between different cell types are probably among the most important mechanisms regulating growth and differentiation in normal embryogenesis.1 When cultured in a nonadhering dish, pluripotent stem cells, such as embryonic stem (ES) cells and embryonic carcinoma (EC) cells, can spontaneously form multicellular aggregates that are similar to postimplantation embryonic tissues in vivo. These cell aggregates are called embryoid bodies (EBs), and they can differentiate into all three germ layers2,3 and form cavitation.4 Since cell aggregation recapitulates many aspects of cell-cell interactions and cell differentiation occurring in early embryogenesis, it has been widely utilized to initiate in vitro differentiation of stem cells. * To whom correspondence should be addressed: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China. Fu-Kun Zhao: tel, +86-21-5492-1159; fax, +86-21-5492-1011; e-mail, fkzhao@ sibs.ac.cn. Nai-He Jing: tel, +86-21-5492-1381; fax, +86-21-5492-1011; e-mail, [email protected]. † Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. ‡ Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. # Zhejiang Sci-Tech University. 10.1021/pr800889p CCC: $40.75

 2009 American Chemical Society

P19 mouse embryonic carcinoma cells are a well-established in vitro model with which to study the molecular mechanisms of early events in neural fate determination and differentiation.5,6 P19 cells differentiate from pluripotent stem cells to neural progenitors when aggregated in nonadherent dishes in the presence of retinoic acid (RA) for 4 days followed by differentiation into neurons and glial cells during subsequent neural differentiation stages.7-9 Cell aggregation is essential for P19 cell neural differentiation. Exposure of monolayer P19 cells to RA leads to the formation of endoderm- and mesodermlike cells,7,8,10 while aggregation of P19 cells without chemical inducers results in the differentiation of extraembryonic endoderm,11,12 suggesting that the neural differentiation of P19 cells requires two separate signals: the RA signal13 and signal(s) from cell aggregation. Conditions that reduce adhesion in cell aggregates inhibit P19 neural differentiation.14 Moreover, our previous studies showed that overexpression of mouse Ncadherin15 and Wnt116 in P19 cells can trigger neural differentiation without RA induction, but cell aggregation was still needed. Furthermore, we recently demonstrated that the expression of fibroblast growth factor 8 (FGF8) is transiently up-regulated upon P19 cell aggregation, and the aggregationdependent FGF signaling is essential for P19 cell neural Journal of Proteome Research 2009, 8, 1765–1781 1765 Published on Web 03/10/2009

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differentiation. These observations suggest that cell aggregation may mediate an independent signaling pathway and play a vital role in the neural differentiation of P19 cells. Similarly, the enhanced cell-cell interaction in EBs is also important for ES cell neural differentiation.18,19 These studies clearly show that cell aggregation is an essential factor for neural differentiation of pluripotent stem cells; however, the precise mechanisms of cell aggregation in this process remain to be elucidated. The proteomics approach is a powerful tool to find clues to investigate the mechanism of a biological process due to its capability of resolving thousands of proteins at one time. The first proteomic analysis of P19 cell aggregation was carried out by Ray and Gottlieb in 1996,20 when they tried to find differentially expressed protein spots between aggregated and nonaggregated P19 cells in their 2DE gels, but they failed to reveal any aggregation-related proteins, probably due to technical limitations. Advances in 2DE technology and peptide mass spectrometry offer a promising proteomic approach to study cell aggregation during P19 cell neural differentiation. This study compares protein expression profiles in aggregated and nonaggregated P19 cells. Since the neural differentiation of Wnt1/P19 cells are cell aggregation dependent, these cells were also used for the proteomic analysis. The differentially expressed proteins in 2DE were then identified by MALDI-TOF/ TOF. We confirmed the reliability of some identified proteins by immunoblot and RT-PCR, and we studied the function of one of the up-regulated proteins by silencing its expression during P19 cell neural differentiation.

Experimental Procedures Cell Culture and Sample Preparation. P19C6, a subclone of the P19 mouse embryonic carcinoma cell line, was used in this study, and the P19 cells were cultured as previously described.15 For collection of nonaggregated cells, P19 and Wnt1/P19 cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) to 70-80% confluence, digested with 0.05% trypsin-0.53 mM EDTA, pelleted by centrifugation at 1000 rpm for 3 min and washed twice with ice-cold PBS (pH 7.2). For collecting aggregated P19 and Wnt1/P19 cells, cells were allowed to aggregate in bacterial grade Petri dishes at a seeding density of 1 × 105 cell/mL in 10% FBS/RMEM (Gibco, Carlsbad, CA). After 4 days of aggregation, cell aggregates were centrifuged at 1000 rpm for 3 min and washed twice with ice-cold PBS. Cell pellets were then lysed in a lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS (w/v), 1% DTT, and 2% IPG (v/v) buffer (pH 3-10 NL)21 for 1 h using a rocker at room temperature and were then centrifuged at 45 000g for 1 h at 4 °C. The supernatant was collected, aliquoted, and then stored at -80 °C for two-dimentional electrophoresis (2DE). Protein concentrations were determined using the Bradford assay.22 Two-Dimensional Electrophoresis (2DE). Two-dimensional electrophoresis was carried out according to the method in the manufacturer’s instructions23 and modified by Gorg et al.21 The IPG strips (24 cm, pH 3-10 NL, GE Healthcare-Amersham Biosciences, Piscataway, NJ) were rehydrated overnight with rehydration buffer (8 M urea, 2% CHAPS, 0.5% IPG buffer, and 0.4% DTT). Three hundred micrograms of protein was loaded on the rehydrated IPG strip via anodic cup loading. IPG-IEF was run on an IPGphor IEF system (GE Healthcare) at 20 °C in five steps: 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h, grandient from 1000 V up to 8000 V in 30 min and 8000 V for 56 000 Vh. After IEF, the IPG strips were immediately equilibrated in 10 1766

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Gao et al. mL of equilibration solutions (6 M urea, 2% SDS (w/v), and 50 mM Tris-HCl buffer at pH 8.8, 30% glycerol (v/v)) containing 0.4% DTT with gentle shaking for 15 min, followed by alkylation with 10 mL of equilibration solutions containing 2.5% iodoacetamide for 15 min. SDS-PAGE was performed on 12.5% polyacrylamide gels (260 mm × 200 mm × 1 mm) with an Ettan DALT twelve apparatus (GE Healthcare). Gels were run at 5 W/gel for 1 h, followed by 20 W/gel until the end of the run. Each group was run at least five times to guarantee reproducibility. After 2DE, the gels were stained with silver nitrate as described by Yan et al.24 Image Acquisition and Statistical Analysis. Silver-stained gels were scanned with a D2000 Uniscan scanner (Tsinghua Uniscan, Beijing, China) in transmissive mode with a resolution of 300 dpi. Spot detection, quantification, and matching were performed with Image Master 2D Platinum Version 5.0 software according to the manufacturer’s instructions (GE Healthcare). For analysis, 3 best gels for each group were chosen. Twosample t tests were used to analyze differences in protein expression between nonaggregated and aggregated P19 or Wnt1/P19 cells. P-value less than 0.05 were considered statistically significant. Digestion of Proteins. All differentially expressed protein spots were manually excised with Spot Picker (1.5 mm, The Gel Company) from the silver-stained gels. These gel spots were digested using the methods of Shevchenko et al.25 and Farzin Gharahdaghi26 with minor modifications. After destaining, washing, and dehydration, the gel spots were incubated with 10 ng/µL sequencing-grade modified trypsin (Promega, Madison, WI) in 25 mM NH4HCO3 at 37 °C for 3 h or overnight. Peptides were sequentially extracted with 5% trifluoroacetic acid (TFA) and 2.5% TFA/50% ceric ammonium nitrate (CAN) by sonication for 3 min. Supernatants were pooled and dried in a Speed-Vac. Peptides were then resuspended in 1.5 µL of 0.5% TFA. Mass Spectrometry and Database Search. Digested peptide samples were cocrystallized with an equal volume of saturated matrix solution R-cyano-4-hydroxycinnamic acid (CHCA) in 50% acetonitrile (ACN)/0.1% TFA on a stainless steel MALDI target plate. MS and MS/MS spectra were obtained using a 4700 proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Foster City, CA). The peaks of the calibration mixture kit (Applied Biosystems, Foster City, CA) in 6 calibration spots were used as external standards to calibrate each spectrum to mass accuracy within 5 ppm in MS and 10 ppm in MS/MS. MS spectra were acquired in MS Reflector Positive Operating Mode, and the peptide masses were acquired for the range from 850 to 4000 m/z with a focus mass of 2000 Da. Four peaks with the strongest signal in MS spectra were automatically selected using an exclusion list of tryptic auto digestion peaks and subjected to MS/MS analysis in MS/MS 1 KV Positive Operating Mode using the interpretation method with the metastable suppressor turned on. Air was used as the collision gas in the collision-induced dissociation (CID). A combined MS and MS/MS search was performed against the Swiss-Prot database release 56.0 of 22-Jul-2008 (392 667 sequence entries) using MASCOT software (Version 2.0; Matrix Science, London, U.K.). The raw MS and MS/MS spectra were processed using GPS Explorer software (Version 3.5; Applied Biosystems) with the following settings: MS peak filtering-mass range, 850-4000 Da; minimum signal-to-noise ratio, 10; peak density filter, 50 peaks per 200 Da; maximum number of peaks, 65; MS/MS peak filtering-mass range, 60-20 Da; minimum

Proteomic Analysis of P19 Cell Aggregation

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Figure 1. Comparison of 2DE patterns between nonaggregated P19 (A) and aggregated P19 cells (B), and nonaggregated Wnt1/P19 (C) and aggregated Wnt1/P19 cells (D). Three hundred micrograms of total protein for each cell status was subjected to 2DE using the cup-loading method (first dimension, IPG strip, pH 3-10 NL, 24 cm; second dimension, 12.5% SDS-PAGE, 260 × 200 × 1 mm3). Proteins were visualized by silver nitrate staining. Differentially expressed proteins are indicated with black circles and numbered.

signal-to-noise ratio, 10; peak density filter, 50 peaks per 200 Da; maximum number of peaks, 65. The searches were conducted using the following settings: taxonomy of Mus, trypsin digestion with one missed cleavage, variable modifications of carbamidomethylation of cysteine and methionine oxidation, peptide mass tolerance of 50 ppm, MS/MS ion tolerance of 0.1 Da. MS spectra with scores over 54 and MS/ MS peptide spectra with ion scores exceeding the threshold were considered as statistically significant search scores (equivalent to >95% confidence interval (C.I.), p < 0.05). Protein scores taken from an MS combined MS/MS search with a minimum C.I. of 95% were defined as positive identifications. All differentially expressed protein spots were identified with MS and MS/MS in triplicates and protein spots with positive identifications and highest scores were selected to report. If a database search matched to multiple proteins and these proteins belong to different protein families, then all the proteins whose Mr and pI also matched to the spot position in 2D-gel were reported and this spot was considered as the protein mixtures. If a database search matched to multiple proteins and these proteins are isoforms, the one with the highest score was selected to report. Protein Classification and Network Analysis. On the basis of expression patterns, proteins were classified as cell aggregation-associated proteins, which differentially expressed in both P19 and Wnt1/P19 cell aggregates, and Wnt signaling-related-

proteins, which differentially expressed only in Wnt1/P19 cells. According to information from KEGG,27 ExPasy (http://expasy.org/sprot/) and literature, aggregation-associated proteins were classified into 10 functional categories. The functional protein association networks and protein interactions were searched by KEGG and STRING database (http://string.embl.de/). The networks were compiled and visualized in Cytoscape2.6.1 software (www.cytoscape.org). Western Blot. The nonaggregated and aggregated P19 and Wnt1/P19 cells were lysed with modified RIPA buffer that contained 50 mM HEPES (pH 7.4), 5 mM EDTA, 50 mM NaCl, 1 mM sodium orthovanadate (Na3VO4), 50 mM sodium fluoride, 1% Triton X-100, 1 mM PMSF, 5 µg/mL aprotinin, and 5 µg/ mL leupeptin. Western blots were performed as described previously.28 Antibodies used for Western blotting included goat antiNDRG1 polyclonal antibody (1:5000, Abcam, Cambridge, MA), rabbit anti-14-3-3 epsilon (T16) polyclonal antibody (1:1000, Santa Cruz, CA), mouse anti-14-3-3 gamma monoclonal antibody, clone CG31-2B6 (1:10 000, Upstate, Lake Placid, NY), rabbit anti-acid calponin polyclonal antibody (1:1500, Abcam, Cambridge, MA), and mouse anti-Cdc2 monoclonal antibody, clone A17 (1:5000, Abcam, Cambridge, MA). The second antibodies were goat anti-mouse, goat anti-rabbit or donkey anti-goat IgG-horseradish peroxidase conjugate (1:10 000, Santa Journal of Proteome Research • Vol. 8, No. 4, 2009 1767

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Figure 2. (A) The magnified comparison maps of the representative proteins from 2DE, including the up-regulated proteins (NDRG1 (spot 15), 14-3-3 epsilon (spot 66)) and the down-regulated proteins (14-3-3 gamma (spot 64), acid calponin (spot 31) and Cdc2 (spot 47)). (B) The statistical analysis of the quantified spots is presented as the % volume of these proteins. N, nonaggregated; A4, aggregated for 4 days.

Cruz, CA). All blots were performed according to the manufacturer’s instructions. RT-PCR. Total RNAs were extracted from nonaggregated and aggregated P19 and Wnt1/P19 cells using Trizol reagent (Invitrogen, Carlsbad, CA). Reverse transcription was performed with 5 µg of total RNA using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). The PCR reaction consisted of denaturation at 94 °C for 45 s, annealing for 45 s, and extension at 72 °C for 45 s. PCR primers and reaction parameters for each gene are shown in Supporting Information Table 1. RNAi, Transfection, and Neural Induction. The vector pPGKsuper was constructed by inserting pGFPN1 with the PGK promoter into pSuper (Invitrogen, Carlsbad, CA), and it was used to express small interfering RNA (siRNA). The target sequence of NDRG1 is 5′-GGAGGAGATACACAACAAC-3′. Empty vector was used as a negative control. Oligonucleotides were synthesized (Sangon, Shanghai, China) and inserted into the pPGKsuper vector in the BglII and Hind III sites. P19 cells were transfected with 3 µg of pPGKsuper or pPGKsuper-siRNA using FuGENE HD Transfection Reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s instructions. The pPGKsuper and the pPGKsuper-siRNA transfected P19 cells were induced by RA for 4 days and differentiated in serumfree N2 medium as previously described.16 P19 cells aggregated 1768


for 2 and 4 days were collected, and total RNA or proteins were extracted for RT-PCR and Western blot analysis, respectively. Immunostaining. Transiently transfected P19 cells were aggregated in the presence of RA for 4 days and then replated in N2 medium for 3 days. Cells were fixed with 4% paraformaldehyde/PBS and immunostained as previously described.15 The following antibodies were used, mouse monoclonal antibodies Oct4 (1:200, Santa Cruz, CA) and Tuj1 (1:200, Sigma, St. Louis, MO), and the rabbit polyclonal antibodies group B1 Sox proteins [Sox1/(2)/3] (1:100).29,30 Fluorescein isothiocyanate (FITC)- and Cy3-conjugated secondary antibodies were obtained from Jackson Immunoresearch laboratories (1:500, West Grove, PA). Normal mouse and rabbit IgGs (Zymed, San Diego, CA) were used as the negative controls. The images were taken with Olympus BX51 fluorescence microscopy (Tokyo, Japan). Statistics. Each experiment was repeated at least three times. Data shown are expressed as mean ( SEM. Student’s t tests were used to compare the effects of all treatments. Differences were considered statistically significant at p < 0.05.

Results Proteomic Profiles of Nonaggregated and Aggregated P19 and Wnt1/P19 Cells. Since both P19 and Wnt1/P19 cells needed to aggregate for 4 days to trigger their neural dif-


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Figure 3. Identification of the NDRG1 protein by MALDI-TOF MS/MS. (A) Peptide mass fingerprints of NDRG1 and the MS/MS spectrum of peptide m/z 1938.86 confirmed from the labeled ions. (B) Amino acid sequences of NDRG1. Matched peptides are underlined. A combined database search of MS and MS/MS measurements was performed, and the information on the identified proteins is shown in Table 1.

ferentiation, we aggregated these cells in the absence of RA for 4 days and collected the nonaggregated and aggregated cells for proteomic analysis. Three batches of cells derived from independent experiments were collected to normalize the effect of variations in the biological replicates. The protein expression profiles were monitored with replicated 2DE. After silver nitrate staining, 2DE images were acquired by the D2000 Uniscan scanner and analyzed by Image Master 2D Platinum Version 5.0 software. More than 3000 protein spots per gel were displayed and the match rate of the spots between any two gels exceeded 80%, suggesting high resolution and reproducibility. Three batches of 2D-profiles with high reproducibility were subjected to statistical analysis. The representative 2DE images are shown in Figure 1, in which the differentially expressed proteins are marked and numbered. Statistical analysis of the normalized quantities of matched spots revealed that 71 protein spots had significantly different expression patterns (>1.5-fold change, p < 0.05, Supporting Information Table 2). Among these 71 protein spots, 54 were differentially expressed in both P19 and Wnt1/P19 cell aggregates, with 42 up-regulated and 12 down-regulated upon aggregation. The other 17 protein spots were differentially expressed only in the Wnt1/P19 cell aggregates, suggesting that these proteins may be involved in Wnt signaling. No proteins were differentially expressed only in the P19 cell aggregates. Five protein spots, numbered 15, 66, 64, 31 and 47, were chosen to validate the differential expression patterns with further study. The magnified images of these spots are shown

in Figure 2A, and the statistical analysis of the quantified spot volumes (% volume) of these proteins are shown in Figure 2B. The distinct expression patterns of these 54 cell aggregationassociated protein spots suggest their possible involvement in the aggregation of P19 cells. Thus, differentially expressed protein spots were isolated for further identification using MALDI-TOF/TOF. Identification of Proteins by MALDI-TOF/TOF and Functional Exploration for Candidate Proteins. To characterize the proteins in the differentially expressed spots by 2DE, all 71 spots were excised from the silver-stained gels and identified by MS and MS/MS. As a representative example, the peptide mass fingerprints and MS/MS spectra of N-myc downstream-regulated gene 1 (NDRG1, spot 15) are illustrated in Figure 3. Detailed information on the 54 proteins associated with P19 cell aggregation is listed in Table 1, including the spot number, protein name (gene name), Swiss-Prot accession number, theoretical Mr (Da)/pI, peptide counts, percentage of sequence coverage, MS scores, sequence confirmation by CID, MS/MS scores and percentage of protein score C.I. The information for the other 17 proteins related to Wnt signaling in Wnt1/P19 cells is also provided in Supporting Information Tables 3 and 4. The 54 cell aggregation-related proteins were classified into 10 functional categories based on the information from KEGG,27 ExPasy (http://www.expasy.org/sprot/) and literature (Table 2 and Figure 4A). These proteins were implicated in a broad range of cellular activities. About 22% of the proteins Journal of Proteome Research • Vol. 8, No. 4, 2009 1769

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Table 1. List of Aggregation-Associated Proteins Identified by MALDI-TOF MS and MS/MS spot no.a

1

protein name (gene name)

Thimet oligopeptidase (Thop1)

Swiss-Prot accession no.

Q8C1A5

theoretical Mr (Da)/pI

peptide sequence MS countc coverage % scored

sequence confirmed by CID

Proteins Up-Regulated in Both P19 Cell and Wnt1/P19 Cell Aggregation 77976.2/5.72 27 36 193 544QANAGLFNLR553

38

100

58 35

100

66 96 120 59

100

67 40 -

59 100

57

100

LSLCGEESFGTGSDHIR LVIGQNGILSTPAVSCIIR104 222 VLATAFDTTLGGR234

87 45 22

100

391

EFSITDVVPYPISLR ALESSIAPIVIFASNR333 185 VEAGDVIYIEANSGAVKR202 340 GTEDITSPHGIPLDLLDR357 306 NLPLPPPPPPR316

18 26 26 39 50

149

LLIHQSLAGGIIGVK163 TDYNASVSVPDSSGPER86 199 EEIHNNVEVVHTYR212

73 37 61

100

560

3

Heat shock protein 75 kDa, mitochondrial precursor (Hsp75)

Q9CQN1

MS/MS protein scoree score C.I.%f

80158.5/6.25

22

45

150

498

VDQVLHTQTDADPAEEYAR NIYYLCAPNR507

578

637

AQLLQPTLEINPR649 DISEFQHEEFYR318 472 YESSALPAGQLTSLPDYASR491 285 CVINASGPFTDSVR298 307

4

Glycerol-3-phosphate dehydrogenase, mitochondrial precursor (Gpd2)

Q64521

80902.5/6.17

24

38

164

270 558

5 6

Heat-shock protein 105 kDa (Hsph1) Phosphoglucomutase-1 (Pgm1)

Q61699

96346.4/5.39

19

25

91

-

Q9D0F9

61478.7/6.30

29

57

269

9

CKDVLTGQEFDVR282 LAFLNVQAAEEALPR572

TQAYPDQKPGTSGLR23

371

387

86

11

Heat shock 70 kDa protein 4 (Hspa4)

Q61316

94073.3/5.15

23

28

140

12

RuvB-like 1 (Ruvbl1)

P60122

50182.3/6.02

19

52

150

14

Heterogeneous nuclear ribonucleoprotein K (Hnrnpk)

P61979

50944.4/5.39

16

45

108

405

318

70

15 16

17

N-myc downstream regulated gene 1 protein (Ndrg1) 2-oxoisovalerate dehydrogenase alpha subunit, mitochondrial precursor (Bckdha) Actin, cytoplasmic 2 (Actg1)

42980.9/5.69

9

37

63

P50136

50339.1/8.14

17

36

122

148

TDLVFGQYR156

42

100

P63260

41765.8/5.31

16

58

139

360

QEYDESGPSIVHR372

64

100

105 70 32 130

100

53 31 81 74

100

86 34 116 18

100

SYELPDGQVITIGNER254 VAPEEHPVLLTEAPLNPK113 292 DLYANTVLSGGTTMYPGIADR312 157 LGDVYVNDAFGTAHR171 96

Phosphoglycerate kinase 1 (Pgk1)

P09411

44522.0/8.02

21

60

175

200

216

ALESPERPFLAILGGAK VLNNMEIGTSLYDEEGAK264 280 ITLPVDFVTADKFDENAK297 85 QLFHPEQLITGK96 247

22

Tubulin alpha-2 chain (Tuba1b)

100

Q62433

239

18

100

P05213

50119.6/4.94

17

53

167

65

AVFVDLEPTVIDEVR79 NLDIERPTYTNLNR229 265 IHFPLATYAPVISAEK280 80 WLAIDANAR88 216

23

Importin beta-1 subunit (Kpnb1)

P70168

97090.1/4.68

12

14

46

26

Proteasome subunit alpha type 3 (Psma3) Heterogeneous nuclear ribonucleoprotein Q (Syncrip) Actin, cytoplasmic 2 (Actg1)

O70435

28387.1/5.29

9

38

58

Q7TMK9

69589.6/8.68

19

33

135

222

LYNNHEIR229

27

100

P63260

41765.8/5.31

14

53

110

360

QEYDESGPSIVHR372

97

100

101 83 39

100

28

28 29

21

AAVENLPTFLVELSR42 VFQVEYAMK29

239

SYELPDGQVITIGNER254 VAPEEHPVLLTEAPLNPK113 169 ADHGEPIGR177 96

34

Heat shock protein HSP 90-beta (Hsp90ab1)

P11499

83273.1/4.97

15

19

78

73

IDILPNPQER82 ELKIDILPNPQER82 181 VILHLKEDQTEYLEER196 96 VAPEEHPVLLTEAPLNPK113 70

36

1770

Actin, cytoplasmic 2 (Actg1)

P63260

41765.8/5.31


11

42

73

27 17

83 40 27 39

99.95

99.94

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Proteomic Analysis of P19 Cell Aggregation Table 1. Continued spot no.a



37 Actin, cytoplasmic 2 (Actg1)

P63260

38 40S ribosomal protein SA (Rpsa)

P14206

39 40S ribosomal protein SA (Rpsa)

P14206


41765.8/5.31

sequence peptide coverage MS c count % scored

10

38

63

sequence confirmed by CID 197 96

32817.4/4.80

7

40

73

90 64

32817.4/4.80

9

44

86

90

GYSFTTTAER206 113

VAPEEHPVLLTEAPLNPK FAAATGATPIAGR102 80

AIVAIENPADVSVISSR FAAATGATPIAGR102

103

117

40 short/branched chain specific Acyl-CoA dehydrogenase, mitochondrial precursor (Acadsb) 41 Actin-like protein 6A (Actl6a)

Q9DBL1

47843.3/8.00

13

37

FTPGTFTNQIQAAFR AIVAIENPADVSVISSR80 FTPGTFTNQIQAAFREPR120 87 409IGTIYEGASNIQLNTIAK426

Q9Z2N8

47417.4/5.39

10

30

58

44 60S acidic ribosomal protein P0 (Rplp0)

P14869

64

103

154 63

34194.8/5.91

13

55

109

67

TAVLTAFANGR164 76

QGGPTYYIDTNALR GHLENNPALEK77

135

146

TSFFQALGITTK AGAIAPCEVTVPAQNTGLGPEK134 AFLADPSAFAAAAPAAAATTAAPAAAAAPKA297 121 85EYPNICAGTDR95 113 267

46 Uridine phosphorylase 1 (Upp1)

P52624

34063.9/6.12

14

58

72

P19096

272256.8/6.13

11

4

49 Electron transfer flavoprotein alpha-subunit, mitochondrial precursor (Etfa)

Q99LC5

34987.5/8.62

15

66

YVAAELGLDHPGK 74 LQGDQINTPHDVLVEYQQRPQR298 VSVHIIEGDHR2475 8 2408 ELSFAAVSFYHK2419 126 102QFSYTHICAGASAFGK117 2465

86

GLLPEELTPLILETQK101 LLYDLADQLHAAVGASR249 124 189GVLHQFSGTETNR201 233

50 Adenylate kinase isoenzyme 4, mitochondrial (Ak3l1) 52 Voltage-dependent anion-selective channel protein 1 (Vdac1)

Q9WUR9 25046.1/7.02

13

68

Q60932

32331.4/8.55

11

47

89

54 26S protease regulatory subunit 8 (Psmc5)

P62196

45597.1/7.11

18

44

140


13

35

41765.8/5.31

10

38

41765.8/5.31

11

45

30 69 71 56

100

30 28 88 113

100 100

60 127 40

100

100

61 110 60 33

100

25 116 22 82

100 100 100

38 36

100

219

25

100

78

20 29

AVFPSIVGRPR39 VAPEEHPVLLTEAPLNPK113 148 TTGIVMDSGDGVTHTVPIYEGYALPHAILR177 73 19AGFAGDDAPR28 206

GYSFTTTAER VAPEEHPVLLTEAPLNPK113

18 10 12

30 34 45

98 75 28AVTEQGAELSNEER41 80 131YLAEFATGNDR141

67 14-3-3 protein beta/alpha Q9CQV8 (Ywhab) 69 Proteasome subunit beta Q60692 type 6 precursor (Psmb6)

28068.9/4.77

9

48

58

30

61

209

72 Ras-GTPase-activating P97855 protein binding protein 1 (G3bp1)

100

100

QEYDESGPSIVHR372 239 SYELPDGQVITIGNER254 93 197GYSFTTTAER206

131

67

YLAEFATGNDRK142 AVTEQGHELSNEER43 LAAIQESGVER

LTPIHDHIFCCR SLDLFGCEVTNR128

117

31060.1/3.89

5

22

30

76676.8/4.69

17

21

51796.8/5.41

10

30

SVSLYYTGEK469 GFGFVDFNSEEDAK621 575 GLSEDTTEETLKESFEGSVR594 60 124FYVHNDIFR132 98

44 49

83 103 23

EELQLLQEQGSYVGEVVR FVVDVDKNIDINDVTPNCR113 LLREELQLLQEQGSYVGEVVR78 94 197GYSFTTTAER206

66744.8/5.11 27760.7/4.96 29155.4/4.63

70 Acidic leucine-rich nuclear Q9EST5 phosphoprotein 32 family, member B (Anp32b) 71 Nucleolin (Ncl) P09405

100

83 102 43 34

78

62 Lamin B1 (Lmnb1) P14733 65 14-3-3 protein tau (Ywhaq) P68254 66 14-3-3 protein epsilon P62259 (Ywhae)

38

20

100

96

8

100

81 59

197

25362.5/4.97

59 53 38 55

EHINLGCDVDFDIAGPSIR152 AVAHHTDCTFIR213

202

29

P63260

100

134

96


53 32

100

360

P63260

100

77

58

41765.8/5.31

79 46

VTQSNFAVGYK187

61

P63260

100

177

95


24

84

277

48 Fatty acid synthase (Fasn)

protein MS/MS score e score C.I.% f

460 608

133

YQDEVFGGFVTEPQEESEEEVEEPEER159

39 102 157 53

98.07

100 100

73

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Table 1. Continued spot no.a

10


Inosine-5′-monophosphate dehydrogenase 2 (Impdh2)


P24547


peptide countc

sequence coverage %

MS scored

sequence confirmed by CID

Proteins Down-Regulated in Both P19 Cell and Wnt1/P19 Cell Aggregation 137 HGFCGIPITDTGR149 55779.8/6.84 20 46 158 209

LPIVNENDELVAIIAR224 GKLPIVNENDELVAIIAR224 270 YITPDQLADLYK281 33 AAVPSGASTGIYEALELR50 257 GMSVYGLGR265 173 CASQAGMTAYGTR185 193 MQTDKPFDQTTISLQMGTNK212 211 YLNFFTK217 207

13

Alpha enolase (Eno1)

P17182

47111.2/6.37

21

60

192

31

Acid calponin (Cnn3)

Q9DAW9

36405.8/5.46

14

48

110

35b

Proliferating cell nuclear antigen (Pcna)

P17918

28766.3/4.66

11

45

84

54

42

Tropomyosin alpha-3 chain (Tpm3) Mitochondrial 39S ribosomal protein L39 (Mrpl39)

P21107

32842.8/4.68

8

24

38

Q9JKF7

38584.7/8.09

17

59

126

92

61

SEGFDTYR IQLVEEELDRAQER105

Thymidylate synthase (Tyms)

P07607

34935.7/6.00

10

37

67

53

Cell division control protein 2 homologue (Cdc2) Phosphoglycerate mutase 1 (Pgam1)

P11440

34085.0/8.39

9

38

94

Q9DBJ1

28813.9/6.67

15

56

151

P14602

22999.7/6.12

12

62

116

64

Peroxiredoxin 6 (Prdx6) Thioredoxin domain containing protein 9 (Txndc9) Glutamatescysteine ligase regulatory subunit (Gclm) 14-3-3 protein gamma (Ywhag)

54 32 72 30

100

60 ---

100

94

100

FSGWYDADLSPAGHEEAK39 NLKPIKPMQFLGDEETVR240 29 LFDQAFGVPR38

105 85 76

100

101

114 134 17

99.99 99.99

18

99.87

22

HGELQYLR29

DFLDSLGFSAR --11

24723.9/5.72 26243.4/5.69

10 10

43 39

80 65

-

O09172

30515.6/5.35

8

35

53

50

28153.9/4.8

15

44

120

120

HGESAWNLENR21

P63028

19449.6/4.76

8

43

71

142

Translationally controlled tumor protein (Tpt1)

VSLDVNHFAPEELTVK116 AVTQSAEITIPVTFEAR192 VIPTLALLR150

TLNEWSSQISPDLVR64

132 28

68

99.99

AFKDDYVVSLVR165 IGDFIDVSEGPLIPR268

O08709 Q9CQ79

P61982

100

154

176

58b

100

100

223

Heat-shock protein beta-1 (Hspb1)

42 50

100

23

22

56b

56 115 55 81 37 33 67 62

100

DLPFETLDVDAR221

110

47

63

protein score C.I.%f

210

254

45

MS/MS scoree

YLAEVATGEKR

142

NVTELNEPLSNEER41 LEEQKPER110

103 101 6

110

GKLEEQKPER DLISHDELFSDIYK19

20

100

85 35

100

52 95

a Spot no. was defined according to spot positions in the 2-D gel indicated as in Figure 1. b For spots 35, 56 and 58, more than one protein was identified from the digested peptides, suggesting that these proteins co-migrated in the gels. c Number of unique peptides matched to mass peaks. d Mascot MS scores were taken from the MS spectra search results using GPS Explorer software (Version 3.5). In this program, a Mascot score >54 was considered significant (p < 0.05). e MS/MS scores were the ion scores taken from the MS/MS peptide spectra search results using GPS Explorer TM software (Version 3.5). f Protein score C.I. percentage based on the MS and MS/MS spectra search.

were related to metabolism, which includes carbohydrate, energy, nucleotide and lipid metabolism. Cytoskeleton (20%) and protein synthesis (16%) accounted for the second and third categories of proteins. Many signal transduction molecules (11%) were also found to be involved in cell aggregation. Meanwhile, a significant number of proteins involved in cell growth (9%), translation (4%), transcription (5%), RNA processing (4%) and transport (2%) were also identified. In addition, we used the combined databases to propose the protein interaction networks (Figure 4B). Nine cell aggregationrelated proteins were correlated to the regulation of cell cycle, gluconeogenesis and proteasome and 21 cell aggregation1772


related proteins were correlated to each other by protein-protein interactions. Validation of Differentially Expressed Proteins by Western Blot and RT-PCR Analysis. To validate the results of the proteomic analysis, five proteins, NDRG1 (spot 15), 14-3-3 epsilon (spot 66), 14-3-3 gamma (spot 64), acid calponin (spot 31) and Cdc2 (spot 47) were chosen to confirm their upregulation or down-regulation by Western blot and RT-PCR analysis (Figure 5). Consistent with proteomic results (Figure 2), Western blots showed that NDRG1 and 14-3-3 epsilon proteins were up-regulated in the aggregated P19 and Wnt1/ P19 cells (A4) compared with their nonaggregated counterparts

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Proteomic Analysis of P19 Cell Aggregation Table 2. Functions of Aggregation-Associated Proteins Identified by MALDI-TOF MS and MS/MS spot no.a

1 3 4 5

classificationb

protein name

Thimet oligopeptidase Heat shock protein 75 kDa, mitochondrial precursor Glycerol-3-phosphate dehydrogenase, mitochondrial precursor Heat-shock protein 105 kDa

functionc

Up-Regulated Proteins in Both P19 Cell and Wnt1/P19 Cell Aggregation Folding, Sorting and Degradation Folding, Sorting and Degradation Metabolism Folding, Sorting and Degradation

6

Phosphoglucomutase-1

Metabolism

11

Heat shock 70 kDa protein 4

Folding, Sorting and Degradation

12

RuvB-like 1

Cell Growth and Death

14

Heterogeneous nuclear ribonucleoprotein K

RNA processing

15

N-myc downstream regulated gene 1 protein

Cell Growth and Death

16

2-oxoisovalerate dehydrogenase alpha subunit, mitochondrial precursor

Transcription

17 18 22 23

Actin, cytoplasmic 2 Phosphoglycerate kinase 1 Tubulin alpha-2 chain Importin beta-1 subunit

Cytoskeleton Metabolism Cytoskeleton Transport

26

Proteasome subunit alpha type 3


28

Heterogeneous nuclear ribonucleoprotein Q

RNA processing

29 34

Actin, cytoplasmic 2 Heat shock protein HSP 90-beta

Cytoskeleton Folding, Sorting and Degradation

36 37 38

Actin, cytoplasmic 2 Actin, cytoplasmic 2 40S ribosomal protein SA

Cytoskeleton Cytoskeleton Others

39 40

40S ribosomal protein SA short/branched chain specific Acyl-CoA dehydrogenase, mitochondrial precursor

Others Metabolism

41

Actin-like protein 6A

Transcription

44

60S acidic ribosomal protein P0

Translation

46

Uridine phosphorylase 1

Metabolism

48

Fatty acid synthase

Metabolism

The peptidase binds 1 zinc ion Chaperone that expresses an ATPase activity Gluconeogenesis and glycerol-3-phosphate metabolic process Prevents the aggregation of denatured proteins in cells under severe stress, on which the ATP levels decrease markedly. Inhibits HSPA8/HSC70 ATPase and chaperone activities. This enzyme participates in both the breakdown and synthesis of glucose. Interacts with TJP1/ZO-1, Phosphorylated upon DNA damage, probably by ATM or ATR. Plays an essential role in oncogenic transformation by MYC and also modulates transcriptional activation by the LEF1/TCF1-CTNNB1 complex and is essential for cell proliferation. One of the major pre-mRNA-binding proteins. May have a growth inhibitory role. Expressed most abundantly in the kidney followed by nervous tissues (hypothalamus, cerebellum, and cerebrum) and the preputial gland. Its expression increases after 13.5 dpc when n-Myc expression is largely down-regulated. Branched chain family amino acid catabolic process and positive regulation of transcription, Structural constituent of cytoskeleton Glycolytic enzyme activity. Major constituent of microtubules. Functions in nuclear protein import, either in association with an adapter protein, like an importin-alpha subunit, which binds to nuclear localization signals (NLS) in cargo substrates, or by acting as autonomous nuclear transport receptor. The subunit of Proteasome which is characterized by its ability to cleave peptides with Arg, Phe, Tyr, Leu, and Glu adjacent to the leaving group at neutral or slightly basic pH. Implicated in mRNA processing mechanisms and Expressed in spinal cord at 14 dpc and onward. structural constituent of cytoskeleton Molecular chaperone, has ATPase activity Structural constituent of cytoskeleton Structural constituent of cytoskeleton Enables malignant tumor cells to penetrate laminin tissue and vessel barriers. Activates precursor thymic anti-OFA/iLRP specific cytotoxic T cell. May induce CD8 T-suppressor cells secreting IL-10. It was originally thought to be a laminin receptor. Has greatest activity toward short branched chain acyl-CoA derivative and straight chain acyl-CoAs and may play a role in controlling the metabolic flux of valproic acid in the development of toxicity of this agent. Involved in transcriptional activation and repression of select genes by chromatin remodeling. It is a translational repressor protein. It controls the translation of the rplJL-rpoBC operon by binding to its mRNA Catalyzes the reversible phosphorolytic cleavage of uridine and deoxyuridine to uracil and ribose- or deoxyribose-1-phosphate. Fatty acid synthetase catalyzes the formation of long-chain fatty acids from acetyl-CoA, malonyl-CoA and NADPH. This multifunctional protein has 7 catalytic activities and an acyl carrier protein.


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Table 2. Continued spot no.a

49

50

52

54 57 59 60 62

65

66 67 69 70

71

72

10

13

31

35

42 45 47

53

1774

protein name

Electron transfer flavoprotein alpha-subunit, mitochondrial precursor

classificationb

The electron transfer flavoprotein serves as a specific electron acceptor for several dehydrogenases, It transfers the electrons to the main mitochondrial respiratory chain via ETF-ubiquinone oxidoreductase Adenylate kinase isoenzyme 4, mitochondrial Metabolism Catalyzes the reaction: ATP + AMP ) 2 ADP and located in Mitochondrion. It expresses in the pyramidal cells in the hippocampus and in the central nervous system in a region-specific manner from the middle stage of embryogenesis to the adulthood in the rodent. Voltage-dependent anion-selective channel protein 1 Signal Transduction Forms a channel through the mitochondrial outer membrane and also the plasma membrane. Related to the behavioral fear response, learning and nerve-nerve synaptic transmission. 26S protease regulatory subunit 8 Folding, Sorting and Involved in the ATP-dependent Degradation degradation of ubiquitinated proteins. Actin, cytoplasmic 2 Cytoskeleton Structural constituent of cytoskeleton Actin, cytoplasmic 2 Cytoskeleton Structural constituent of cytoskeleton Actin, cytoplasmic 2 Cytoskeleton Structural constituent of cytoskeleton Lamin B1 Cytoskeleton Components of the nuclear lamina which is thought to provide a framework for the nuclear envelope and may also interact with chromatin. 14-3-3 protein tau Signal Transduction Adapter protein implicated in the regulation of a large spectrum of both general and specialized signaling pathway 14-3-3 protein epsilon Signal Transduction Protein domain specific binding 14-3-3 protein beta/alpha Signal Transduction Protein domain specific binding Proteasome subunit beta type 6 precursor Folding, Sorting and Cleavage of peptide bonds with very Degradation broad specificity Acidic leucine-rich nuclear phosphoprotein 32 family, member B Cell Growth and Death Multifunctional protein working as a cell cycle progression factor as well as a cell survival factor. Required for the progression from the G1 to the S phase. Antiapoptotic protein which functions as a caspase-3 inhibitor. Nucleolin Transcription It is the major nucleolar protein of growing eukaryotic cells. May play a role in the process of transcriptional elongation Ras-GTPase-activating protein binding protein 1 Signal Transduction May be a regulated effector of stress granule assembly. Play a role in the RNA-protein complex mediated transport of tau mRNA from neuronal cell body to the distal axon in developing neurons Down-Regulated Proteins in Both P19 Cell and Wnt1/P19 Cell Aggregation Inosine-5′-monophosphate dehydrogenase 2 Metabolism Rate limiting enzyme in the de novo synthesis of guanine nucleotides and therefore is involved in the regulation of cell growth. It may also have a role in the development of malignancy and the growth progression of some tumors. Alpha enolase Metabolism Plays a part in various processes such as glycolysis, growth control, hypoxia tolerance and allergic responses. May also function in the intravascular and pericellular fibrinolytic system. Acid calponin Cytoskeleton Thin filament-associated protein that is implicated in the regulation and modulation of smooth muscle contraction. Proliferating cell nuclear antigen Cell Growth and Death This protein is an auxiliary protein of DNA polymerase delta and is involved in the control of eukaryotic DNA replication by increasing the polymerase’s processibility during elongation of the leading strand. Tropomyosin alpha-3 chain Cytoskeleton Binds to actin filaments in muscle and nonmuscle cells. Plays a central role, in association with the troponin complex, in the calcium dependent regulation of vertebrate striated muscle contraction Mitochondrial 39S ribosomal protein L39 Translation Mitochondrial genome maintenance Thymidylate synthase Metabolism Nucleotide and deoxyribonucleotide biosynthesis Cell division control protein 2 homologue Cell Growth and Death Plays a key role in the control of the eukaryotic cell cycle. It is required in higher cells for entry into S-phase and mitosis. Phosphoglycerate mutase 1 Metabolism Energy production and utilization, a role in METH-induced dopaminergic neurotoxicity


Metabolism

functionc

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Proteomic Analysis of P19 Cell Aggregation Table 2. Continued spot no.a

56

58

classificationb

protein name

functionc

Heat-shock protein beta-1


Peroxiredoxin 6


Thioredoxin domain containing protein 9

Others

Glutamate-cysteine ligase regulatory subunit

Metabolism

64

14-3-3 protein gamma

Signal Transduction

68

Translationally controlled tumor protein

Others

a Spot no. was defined according to spot positions in the 2-D gel indicated as in Figure 1. ExPasy 3Hand literature. c Protein functions adapted from http://www.us.expasy.org/sprot/.

(N) (Figure 5A). The down-regulated proteins, 14-3-3 gamma, acid calponin and Cdc2, were also confirmed by Western blot analysis. RT-PCR showed that NDRG1 mRNA was up-regulated after both P19 and Wnt1/P19 cell aggregation, while 14-3-3 gamma and acid calponin mRNA were down-regulated in the aggregated P19 and Wnt1/P19 cells (Figure 5B). These results showed consistency between mRNA and protein expression patterns for these proteins. However, there was no difference in 14-3-3 epsilon mRNA expression levels between aggregated P19 and Wnt1/P19 cells and their nonaggregated counterparts. The Cdc2 mRNA decreased dramatically in aggregated P19 cells (A4) but showed no change in expression between aggregated and nonaggregated Wnt1/P19 cells (Figure 5B). This inconsistency between mRNA and protein expression suggests that the 14-3-3 epsilon and Cdc2 protein may be regulated by translational/post-translational mechanisms. Inhibition of NDRG1 Expression by RNA Interference Leads to Impaired Cell Aggregation during RA-Induced P19 Cell Neural Differentiation. Our previous data showed that NDRG1 protein levels were significantly increased during P19 cell aggregation. To investigate the function of this protein during RA-induced P19 cell neural differentiation, we analyzed NDRG1 expression by RT-PCR and found that NDRG1 is not expressed either in noninduced P19 cells (N) or on the first day of aggregation and neural induction (A1). The NDRG1 mRNA began to be expressed on the second day of neural induction, and its expression gradually increased with the highest expression on day 4 of cell aggregation in the presence of RA (Figure 6A). RNA interference (RNAi) was used to confirm the function of NDRG1 in P19 cell aggregation. The NDRG1 RNAi expression vector (siRNA) was transiently transfected into P19 cells, followed by aggregation in the presence of RA for 4 days. The empty RNAi vector (pPGKsuper) served as the negative control. The expression of NDRG1 was markedly down-regulated at both the protein (Figure 6B) and mRNA (Figure 6C) levels at day 4 of P19 cell aggregation in the presence of RA. Knock-down of NDRG1 expression impaired P19 cell aggregation and showed much smaller cell aggregates compared with the control cells at day 2 (Agg D2) and day 4 (Agg D4) of P19 cell neural differentiation (Figure 6E). To quantify the size differences of the cell aggregates between the siRNA and control P19 cells, we measured the diameters of cell

b

Involved in stress resistance and actin organization. Involved in redox regulation of the cell, May play a role in the regulation of phospholipid turnover as well as in protection against oxidative injury. Not known, associated with cell differentiation. Glutamate-cysteine ligase catalytic subunit binding Adapter protein implicated in the regulation of a large spectrum of both general and specialized signaling pathway. Binds to a large number of partners, usually by recognition of a phosphoserine or phosphothreonine motif. Binding generally results in the modulation of the activity of the binding partner. Involved in calcium binding and microtubule stabilization

Functional classification based on information from KEGG,

aggregates and plotted them against the percentage of the total number of cell aggregates (Figure 6F). On day 2 of P19 cell aggregation, the highest percentage of siRNA treated cell aggregates had a diameter in the range of 100-125 µm, while the highest percentage of control cell aggregates had a diameter of 175-200 µm (upper panel, Figure 6F). Similarly, on day 4 of P19 cell aggregation, the highest percentage of siRNA cell aggregates had a diameter of 175-200 µm, while the control cells had a diameter of 225-250 µm (lower panel, Figure 6F). N-cadherin, a cell adhesion molecule, plays an important role in cell-cell interactions and P19 cell neural differentiation.15 To dissect the mechanism(s) underlying the relationship between NDRG1 knock-down and cell aggregation impairment, we examined N-cadherin expression in NDRG1 RNAi and control P19 cells and found that knock-down of NDRG1 expression indeed decreased the expression of N-cadherin (Figure 6D), suggesting that N-cadherin may mediate the function of NDRG1 in P19 cell aggregation. Inhibition of NDRG1 Expression Results in Impaired P19 Cell Neural Differentiation. Given that P19 cell neural differentiation is dependent on cell aggregation15-17,19 and NDRG1 knock-down impairs P19 cell aggregation, we wondered whether inhibition of NDRG1 expression could affect P19 cell neural differentiation. To confirm this notion, we inhibited NDRG1 expression with RNAi and induced P19 cells to differentiate into neural stem cells by aggregation for 4 days in the presence of RA. The cell aggregates were then doublestained with Oct4 and Sox antibodies (Figure 7A). The noninduced P19 cells expressed both the pluripotent stem cell marker Oct431 and Sox proteins, while the RA-induced P19 cells ceased Oct4 expression but continued to express Sox proteins.17,32 These Oct4-negative and Sox-positive (Oct4-Sox+) cells were defined as neural stem cells that are capable of generating three types of neural cell lineages.33 Inhibition of NDRG1 reduced Oct4-Sox+ neural stem cells (Figure 7B). To further elucidate the effects of NDRG1 silencing, we dissociated the cell aggregates into a single cell suspension and replated them onto cell culture dishes in serum-free N2 medium. Type III β-tubulin (Tuj1) was used as a neuronal marker. Immunostaining showed that inhibition of NDRG1 expression by RNAi reduced Tuj1positive cells on the third day of the neuronal differentiation of P19 cells (Figure 7C,D). Taken together, NDRG1 expression Journal of Proteome Research • Vol. 8, No. 4, 2009 1775

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Figure 4. Functional characterization of differentially expressed proteins related to P19 cell aggregation. (A) Functional classification based on information from KEGG, ExPasy 2Hand literature annotation. (B) Proposed interaction network of 30 proteins related to P19 cell aggregation. The interaction network includes both KEGG pathway (blue line) and direct protein interactions base on information from STRING database (red line). The pink spots indicated proteins that up-regulated in aggregation, the green spots indicated proteins that down-regulated in aggregation, and the blue spots indicated proteins that were involved in the pathways but not identified in this study.

in aggregated P19 cells was necessary for P19 cell neural differentiation.

Discussions Cell-cell interactions are enhanced in cell aggregation, and they are a critical precondition for neural differentiation of P19 cells. Traditional procedures and molecular biological methods contribute to our understanding of the molecular mechanisms involved in cell aggregation during pluripotent stem cell differentiation, such as cadherins,15 the RhoA/Rock pathway34 and the FGF pathway.17 However, little is known about the changes in cellular context that occur during P19 cell aggregation, which are important for subsequent neural differentiation. Using a high-throughput proteomics approach to investigate proteome changes during cell aggregation is necessary and of great importance to further understand the molecular mechanisms involved. 1776


We previously demonstrated that proteomic tools are valuable in studying the differentially expressed proteins in RAinduced P19 cell neural differentiation.35 The present study combined 2DE with MALDI-TOF/TOF to identify proteins with differential expression patterns in cell aggregation. Wild-type P19 and Wnt1/P19 cells16 were subjected to proteomic analysis. The 54 cell aggregation-associated proteins that were identified in both cell lines are listed in Table 1. Another 17 proteins, which were only differentially expressed in Wnt1/P19 cells, were also identified (Supporting Information Table 3). Moreover, the up-regulated NDRG1 and 14-3-3 epsilon proteins as well as the down-regulated 14-3-3 gamma, acid calponin and Cdc2 proteins were confirmed by immunoblotting during P19 cell aggregation (Figure 5). RT-PCR results of NDRG1, 14-3-3 gamma and acid calponin were consistent with 2D results. But there was no difference in 14-3-3 epsilon mRNA level, while the 2D and Western blot results showed the up-regulation of


research articles

Figure 5. Verification of 2DE results. (A) Immunoblot and (B) RT-PCR were used to confirm the expression pattern of NDRG1, 14-3-3 epsilon, 14-3-3 gamma, acid calponin and Cdc2 proteins in the nonaggregated (N) and aggregated (A4) P19 and Wnt1/P19 cells.

14-3-3 epsilon protein levels during P19 cell aggregation. This inconsistency between mRNA and protein expression may be caused by translational regulation or post-translational modification. We did not find the isoforms of 14-3-3 epsilon in 2D gel, it may be because the amount of the isoforms was too small to be detectable or the inconsistency was caused by translation regulation. The cell aggregation-associated proteins found in this study also showed similar expression patterns in other pluripotent stem cell differentiation systems. For example, the downregulation of 14-3-3 gamma protein was also observed during cell aggregation during ES cell differentiation.36 The expression pattern changes for inosine-5′-monophosphate dehydrogenase 2, uridine phosphorylase 1 and heat-shock protein beta-1 in P19 cell aggregation (Table 1) were similar to neural differentiation of mouse ES cells,37 suggesting the possible involvement of these aggregation-associated proteins during pluripotent stem cell differentiation. Functional classification of the differentially expressed proteins showed that almost a quarter of the cell aggregationassociated proteins were related to carbohydrate, energy, nucleotide and lipid metabolism (Figure 4), indicating that cellular metabolic activities are enhanced during P19 cell neural differentiation. Another major component of the identified proteins is the cytoskeleton proteins. Actin (cytoplasmic 2) was presented as multiple protein spots that had different molecular weights and/or pI’s. This variation can be attributed to the degradation or post-translational modification of actins, which might have functions in neural differentiation. This group encompassed not only constitutive proteins involved in the cellular cytoskeleton, such as actin and tubulin, but also proteins involved in modifying and rearranging the cytoskeleton, such as acid calponin, tropomyosin 1 alpha chain and lamin B1 (Table 2). These results suggest that cytoskeletal arrangement may be required during cell aggregation and the neural differentiation process to define the morphological properties of the newly generated neural cells. In this study, spots 38 and 39 had similar molecular weights but different pI’s, but both were identified as 40S ribosomal protein SA (Figure 1 and Table 1), suggesting that this protein may undergo post-translational modifications. This protein is

known to be a non-integrin-type laminin receptor, a component of the small ribosomal subunit that belongs to the ribosomal protein S2P family and may be involved in cell differentiation,38 growth, apoptosis39 and cancer invasion.40 40S ribosomal protein SA was highly expressed in the ES cellderived neural stem cells.41 This result implies that both 40S ribosomal protein SA and its modified form may play important roles in P19 cell aggregation. Cell cycle exit represents the fundamental step to trigger differentiation of cells. In our study, we have found several proteins involved in cell cycle including Pcna, Cdc2, 14-3-3 gamma and Impdh2 (Figure 4B). They were all down-regulated during P19 cell aggregation, indicating cessation of cell cycle and the initiation of P19 cell neural differentiation. Heat shock proteins (HSP) are known to function in protein folding, sorting and degradation. Several HSPs were also identified as cell aggregation-associated proteins, including HSP75(Trp1),HSP105(Hsph1),HSP70(Hspa4),HSP84(Hsp90ab1), and HSP27 (Hspb1). Some have also been identified in other proteomics studies related to the mouse ES cell differentiation.37,42 In addition to their roles in adaptation to stress, heat shock proteins are also involved in different steps during stem cell differentiation and development.43 However, the function and mechanism of the involvement of heat shock proteins in neural differentiation remain unknown and need further investigation. Signal transduction pathways are known to play key roles in early embryogenesis and cell fate decisions,1 and we identified many proteins that may be involved in signaling pathways. The 14-3-3 family members are intracellular dimeric adapter proteins, which play a central role in many cellular processes, including signal transduction, cell cycle, and apoptotic pathways, by modulating the activities of Raf, PKC, and other kinases.44,45 These proteins also have functions in neurogenesis46 and neuronal migration.47 This study showed that during P19 cell aggregation 14-3-3 epsilon was up-regulated while 143-3 gamma was down-regulated (Figure 5). Since it has been reported that different 14-3-3 isoforms have different functions in Xenopus development,48 we presume that the 14-3-3 isoforms with different expression patterns during P19 cell differentiation may have separate functions in neural differentiation. Journal of Proteome Research • Vol. 8, No. 4, 2009 1777

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Figure 6. Inhibition of NDRG1 expression by RNAi impaired P19 cell aggregation. (A) RT-PCR analysis of NDRG1 expression during RA-induced P19 cell neural differentiation. Days after cell aggregation were named as A1, A2, A3, and A4. N: nonaggregated P19 cells. (B) Immunoblot and (C) RT-PCR analysis confirmed the inhibition of NDRG1 expression by siRNA during RA-induced P19 cell neural differentiation. P19 cells were transiently transfected with NDRG1 RNAi vector (siRNA) and the control RNAi vector (pPGKsuper) and were aggregated in the presence of RA for 4 days. The protein and mRNA were extracted from P19 cells at day 2 (A2) and day 4 (A4) of neural differentiation. (E) The cell aggregate size of NDRG1 siRNA cells was smaller than that of the control cells (pPGKsuper) at day 2 (Agg D2) and day 4 (Agg D4) of P19 cell neural differentiation. Scale bar: 100 µm. (F) Quantitative analysis of the diameters of siRNA and pPGKsuper P19 cell aggregates at day 2 (Agg D2) and day 4 (Agg D4) of P19 cell neural differentiation. (D) RT-PCR analysis showed that N-cadherin expression was decreased in the siRNA P19 cell aggregates compared with the control cell (pPGKsuper) aggregates.

The intracellular protein NDRG1 has been studied as a protein associated with stress response,49,50 cell growth,51 carcinogenesis52,53 and differentiation. Induction of NDRG1 expression is associated with the processes of cell differentiation, such as trophoblast recovery from hypoxia-induced injury,54 mast cell maturation55 and tumor cell differentiation.56,57 During mouse early development, NDRG1 is expressed uniformly throughout the extraembryonic ectoderm at embryonic day (E) 5.5-7.5 and is expressed in the node of E7.5 and E8.0 embryos.58 It has also been reported that a nonsense mutation in the NDRG1 gene causes hereditary motor and sensory neuropathy, such as Charcot-Marie-Tooth disease type 4D.59 NDRG1-deficient mice exhibit peripheral nerve degeneration caused by severe demyelination, but complicated motor abilities are retained.60 Therefore, this protein has been categorized as a differentiation-regulated gene, and it might be involved in cell differentiation. In this study, we also observed that NDRG1 was up-regulated in aggregation and RA-induced P19 1778


cell neural differentiation (Figure 5). NDRG1 was chosen as a candidate protein to investigate the mechanism of cell aggregation during P19 cell neural differentiation. We found that NDRG1 up-regulation is cell aggregation associated, and silencing of NDRG1 leads to impaired formation of P19 cell aggregates. This might be due to the impaired cell-cell interactions, since expression of the cell adhesion molecule N-cadherin was inhibited in the NDRG1 knock-down P19 cells (Figure 6). Consistent with these results, we also found that silencing of NDRG1 expression could impair P19 cell neural differentiation (Figure 7). On the basis of these observations, we speculate that NDRG1 may be related to cell adhesion and the cell-cell interactions needed for P19 cell aggregation during neural differentiation. In conclusion, this study provides useful information on the involvement of cell aggregation during P19 cell neural differentiation, and it will facilitate further investigations on the

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Figure 7. Inhibition of NDRG1 expression impaired the neural differentiation of P19 cells. (A) Double staining of Oct4 and Sox proteins from pPGKsuper and siRNA transfected P19 cell aggregates. (B) Percentage of Oct4-Sox+ neural stem cells under the above conditions. (C) Immunostaining of Tuj1-positive neurons after replating P19 cell derived neural stem cells in N2 medium for 3 days. (D) Percentage of Tuj1+ neurons under the above conditions. Scale bar: 50 µm.

relationship between cell-cell interactions and pluripotent stem cell neural differentiation.

Acknowledgment. We thank Mr. Kehua Zhang for the help with constructing siRNA plasmids, Mr. Jianfeng Lin for the help with 2-DE and Mr. Chaochao Wu for the help with pathway network analysis. This work was supported in part by the Creation Foundation from the Shanghai Institutes for Life Sciences and the National Natural Science Foundation of China (30623003, 3072105, 30830034), National Key Basic Research and Development Program of China (2005CB522704, 2006CB943902, 2007CB947101, 2008KR069, 2009CB941100), National High-Tech Research and Development Program of China (2006AA02Z186), Shanghai Key Project of Basic Science Research (06DJ14001, 06DZ22032, 08DJ1400501) and the Council of Shanghai Municipal Government for Science and Technology (088014199).

Supporting Information Available: Primer lists and reaction parameters for RT-PCR are given in supplementary Table 1. Statistical analysis of the quantified spot volumes of differentially expressed proteins is given in supplementary Table 2. The identification information and functional exploration of differentially expressed only in Wnt1/P19 cell aggregation is given in supplementary Tables 3 and 4. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Tam, P. P.; Loebel, D. A. Gene function in mouse embryogenesis: get set for gastrulation. Nat. Rev. Genet. 2007, 8 (5), 368–81. (2) Martin, G. R.; Evans, M. J. Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro. Proc. Natl. Acad. Sci. U.S.A. 1975, 72 (4), 1441–5. (3) Desbaillets, I.; Ziegler, U.; Groscurth, P.; Gassmann, M. Embryoid bodies: an in vitro model of mouse embryogenesis. Exp. Physiol. 2000, 85 (6), 645–51.


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Comparative Proteomic Analysis of Proteins Involved in Cell

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