Rabbit embryonic stem (rES) cells can be derived from various sources of embryos. However, understanding of the gene expression profile, which distincts embryonic stem (ES) cells from other cell types, is still extremely limited. In this study, we compared the protein profiles of three independent lines of rabbit cells, i.e., fibroblasts, fertilized embryo-derived stem (f-rES) cells, and parthenote-derived ES (p-rES) cells. Proteomic analyses were performed using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Collectively, the expression levels of 100 out of 284 protein spots differed significantly among these three cell types (p<0.05). Of those differentially expressed spots, 91% were identified in the protein database and represented 63 distinct proteins. Proteins with known identities are mainly localized in the cytoplasmic compartments (48%), nucleus (14%), and cytoskeletal machineries (13%). These proteins were majorly involved in biological functions of energy and metabolic pathways (25%), cell growth and maintenance (25%), signal transduction (14%), and protein metabolisms (10%). When protein expression levels among cell types were compared, six proteins associated with a variety of cellular activities, including structural constituents of the cytoskeleton (tubulins), structural molecule (KRT8), catalytic molecules (α-enolase), receptor complex scaffold (14-3-3 protein sigma), microfilament motor proteins (Myosin-9), and heat shock protein (HSP60), were found highly expressed in p-rES cells. Two proteins related to HSP activity and structural constituent of cytoskeleton in f-rES cells, and one structural molecule activity protein in fibroblasts showed significantly higher expression levels (p<0.05). Marker protein expressions in f-rES and p-rES cells were further confirmed by Western blotting and immunocytochemical staining. This study demonstrated unique proteomic profiles of the three rabbit cell types and revealed some novel proteins differentially expressed between f-rES and p-rES cells. These analyses provide insights into rES cell biology and would invite more in-depth studies toward rES cell applications.
References
[1]
Graves KH, Moreadith RW (1993) Derivation and characterization of putative pluripotential embryonic stem cells from preimplantation rabbit embryos. Mol Reprod Dev 36: 424–433.
[2]
Schoonjans L, Albright GM, Li JL, Collen D, Moreadith RW (1996) Pluripotential rabbit embryonic stem (ES) cells are capable of forming overt coat color chimeras following injection into blastocysts. Mol Reprod 45: 439–443.
[3]
Fang ZF, Gai H, Huang YZ, Li SG, Chen XJ, et al. (2006) Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos. Exp Cell Res 312: 3669–3682.
[4]
Wang S, Tang X, Niu Y, Chen H, Li B, et al. (2007) Generation and characterization of rabbit embryonic stem cells. Stem Cells 25: 481–489.
[5]
Honda A, Hirose M, Inoue K, Ogonuki N, Miki H, et al. (2008) Stable embryonic stem cell lines in rabbits: potential small animal models for human research. Reprod Biomed Online 17: 706–715.
[6]
Catunda AP, Gócza E, Carstea BV, Hiripi L, Hayes H, et al. (2008) Characterization, chromosomal assignment, and role of LIFR in early embryogenesis and stem cell establishment of rabbits. Cloning Stem Cells 10: 523–534.
[7]
Chiang SK, Chang HH, Ou YW, Intawicha P, Cheng SP, et al. (2008) Successful induction of antisera against rabbit embryos for isolation of the ICM and putative embryonic stem cells. Reprod Domest Anim 43: 181–188.
[8]
Intawicha P, Ou YW, Lo NW, Zhang SC, Chen YZ, et al. (2009) Characterization of embryonic stem cell lines derived from New Zealand White Rabbit embryos. Cloning Stem Cells 11: 27–38.
[9]
Xue F, Ma Y, Chen YE, Zhang J, Lin TA, et al. (2012) Recombinant rabbit leukemia inhibitory factor and rabbit embryonic fibroblasts support the derivation and maintenance of rabbit embryonic stem cells. Cell Reprogram 14: 364–376.
[10]
Fundele R, Norris ML, Barton SC, Fehlau M, Howlett SK, et al. (1990) Temporal and spatial selection against parthenogenetic cells during development of fetal chimeras. Development 108: 203–211.
[11]
Kono T, Obata Y, Wu Q, Niwa K, Ono Y, et al. (2004) Birth of parthenogenetic mice that can develop to adulthood. Nature 428: 860–864.
[12]
Hsieh YC, Intawicha P, Lo NW, Lee KH, Ju JC (2011) LIF and FGF cooperatively support stemness of rabbit embryonic stem cells derived from parthenogenetically activated embryos. Cell Reprogram 13: 241–255.
[13]
Gong SP, Lee EJ, Lee ST, Kim H, Lee SH, et al. (2008) Improved establishment of autologous stem cells derived from preantral follicle culture and oocyte parthenogenesis. Stem Cells Dev 17: 695–712.
[14]
Prudhomme W, Daley GQ, Zandstra P, Lauffenburger DA (2004) Multivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation signaling. Proc Natl Acad Sci U S A 101: 2900–2905.
[15]
Wang D, Gao L (2005) Proteomic analysis of neural differentiation of mouse embryonic stem cells. Proteomics 5: 4414–4426.
[16]
Guo X, Ying W, Wan J, Hu Z, Qian X, et al. (2001) Proteomic characterization of early-stage differentiation of mouse embryonic stem cells into neural cells induced by all-trans retinoic acid in vitro. Electrophoresis 22: 3067–3075.
[17]
Wen J, Xia Q, Lu C, Yin L, Hu J, et al. (2007) Proteomic analysis of cardiomyocytes differentiation in mouse embryonic carcinoma P19CL6 cells. J Cell Biochem 102: 149–160.
[18]
Li Y, Kang X, Guo K, Li X, Gao D, et al. (2009) Proteome alteration of early-stage differentiation of mouse embryonic stem cells into hepatocyte-like cells. Electrophoresis 30: 1431–1440.
[19]
Nasrabadi D, Rezaei Larijani M, Pirhaji L, Gourabi H, Shahverdi A, et al. (2009) Proteomic analysis of monkey embryonic stem cell during differentiation. J Proteome Res 8: 1527–1539.
[20]
Williamson AJ, Smith DL, Blinco D, Unwin RD, Pearson S, et al. (2008) Quantitative proteomics analysis demonstrates post-transcriptional regulation of embryonic stem cell differentiation to hematopoiesis. Mol Cell Proteomics 7: 459–472.
[21]
Jadaliha M, Lee HJ, Pakzad M, Fathi A, Jeong SK, et al.. (2012) Quantitative proteomic analysis of human embryonic stem cell differentiation by 8-plex iTRAQ labeling. PLoS One 7: e38532. Available: http://www.plosone.org/article/info:doi/？10.1371/journal.pone.0038532. Accessed 2013 Jun 11.
[22]
G？rg A, Weissn W, Dunn MJ (2004) Current state of two-dimensional electrophoresis technology for proteomics. Proteomics 4: 3665–3685.
[23]
Huang SY, Lin JH, Chen YH, Chuang CK, Lin EC, et al. (2005) A reference map and identification of porcine testis proteins using 2-DE and MS. Proteomics 5: 4205–4212.
[24]
Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9: 255–262.
[25]
Huang SY, Chen YH, Teng SH, Chen IC, Ho LL, et al. (2006) Protein expression of lymphocytes in HLA-DR transgenic pigs by a proteomic approach. Proteomics 6: 5815–5825.
[26]
Havlis J, Thomas H, Sebela M, Shevchenko A (2003) Fast-response proteomics by accelerated in-gel digestion of proteins. Anal Chem 75: 1300–1306.
[27]
Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20: 3551–3567.
[28]
SAS Institute, Statistical Analysis System (SAS) (2002) User’s Guide: statistics Release 9.1 ed., SAS Institute Inc., Cary, NC.
[29]
Kaufman MH, Barton SC, Surani MA (1977) Normal postimplantation development of mouse parthenogenetic embryos to the forelimb bud stage. Nature 265: 53–55.
[30]
Surani MA, Barton SC, Norris ML (1984) Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 308: 548–550.
[31]
Bouwens L (1998) Cytokeratins and cell differentiation in the pancreas. J Pathol 184: 234–239.
[32]
Nogales E (2001) Structural insight into microtubule function. Annu Rev Biophys Biomol Struct 30: 397–420.
[33]
Arner A, L？fgren M, Morano I (2003) Smooth, slow and smart muscle motors. J Muscle Res Cell Motil 24: 165–173.
[34]
Perez EA (2009) Microtubule inhibitors: Differentiating tubulin-inhibiting agents based on mechanisms of action, clinical activity, and resistance. Mol Cancer Ther 8: 2086–2095.
[35]
Li JY, Wu CF (2003) Perspectives on the origin of microfilaments, microtubules, the relevant chaperonin system and cytoskeletal motors–a commentary on the spirochaete origin of flagella. Cell Res 13: 219–227.
[36]
Attard G, Greystoke A, Kaye S, De Bono J (2006) Update on tubulin-binding agents. Pathol Biol 54: 72–84.
[37]
Andre N, Meille C (2006) Taxanes in paediatric oncology: and now? Cancer Treat Rev 32: 65–73.
[38]
Pasquier E, Kavallaris M (2008) Microtubules: a dynamic target in cancer therapy. IUBMB Life 60: 165–170.
[39]
Wang D, Gao L (2005) Proteomic analysis of neural differentiation of mouse embryonic stem cells. Proteomics 5: 4414–4426.
[40]
Sternlicht H, Farr GW, Sternlicht ML, Driscoll JK, Willison K (1993) The t-complex polypeptide 1 complex is a chaperonin for tubulin and actin in vivo. Proc Natl Acad Sci U S A 90: 9422–9426.
[41]
Gao Y, Thomas JO, Chow RL, Lee GH, Cowan NJ (1992) A cytoplasmic chaperonin that catalyzes beta-actin folding. Cell 69: 1043–1050.
[42]
Yaffe MB, Farr GW, Miklos D, Horwich AL, Sternlicht ML, et al. (1992) TCP1 complex is a molecular chaperone in tubulin biogenesis. Nature 358: 245–248.
[43]
Roobol A, Holmes FE, Hayes NV, Baines AJ, Carden MJ (1995) Cytoplasmic chaperonin complexes enter neurites developing in vitro and differ in subunit composition within single cells. J Cell Sci 108: 1477–1488.
[44]
Mu？oz IG, Yébenes H, Zhou M, Mesa P, Serna M, et al. (2011) Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin. Nat Struct Mol Biol 18: 14–19.
[45]
Yokota S, Yanagi H, Yura T, Kubota H (1999) Cytosolic chaperonin is up-regulated during cell growth. Preferential expression and binding to tubulin at G (1)/S transition through early S phase. J Biol Chem 274: 37070–37078.
[46]
Gupta MK, Jang JM, Jung JW, Uhm SJ, Kim KP, et al. (2009) Proteomic analysis of parthenogenetic and in vitro fertilized porcine embryos. Proteomics 9: 2846–2860.
[47]
Chae HZ, Kim HJ, Kang SW, Rhee SG (1999) Characterization of three isoforms of mammalian peroxiredoxin that reduce peroxides in the presence of thioredoxin. Diabetes Res Clin Pract 45: 101–112.
[48]
Leyens G, Donnay I, Knoops B (2003) Cloning of bovine peroxiredoxins-gene expression in bovine tissues and amino acid sequence comparison with rat, mouse and primate peroxiredoxins. Comp Biochem Physiol B Biochem Mol Biol 136: 943–955.
[49]
Kubo E, Singh DP, Fatma N, Akagi Y (2009) TAT-mediated peroxiredoxin 5 and 6 protein transduction protects against high-glucose-induced cytotoxicity in retinal pericytes. Life Sci 84: 857–864.
[50]
Kim SH, Fountoulakis M, Cairns N, Lubec G (2001) Protein levels of human peroxiredoxin subtypes in brains of patients with Alzheimer’s disease and Down syndrome. J Neural Transm Suppl 61: 223–235.
[51]
Hansen JM, Moriarty-Craige S, Jones DP (2007) Nuclear and cytoplasmic peroxiredoxin-1 differentially regulate NF-kappa B activities. Free Radic Biol Med 43: 282–288.
[52]
O’Flaherty C, de Souza AR (2011) Hydrogen peroxide modifies human sperm peroxiredoxins in a dose-dependent manner. Biol Reprod 84: 238–347.
[53]
Lee YI, Kang WD, Kim MY, Cho MK, Chun SY (2011) Expression of peroxiredoxin I regulated by gonadotropins in the rat ovary. Clin Exp Reprod Med 38: 18–23.
[54]
Di Quinzio MK, Oliva K, Holdsworth SJ, Ayhan M, Walker SP, et al. (2007) Proteomic analysis and characterisation of human cervico-vaginal fluid proteins. Aust N Z J Obstet Gynaecol 47: 9–15.
[55]
Wang S, Huang W, Shi H, Lin C, Xie M, et al. (2010) Localization and expression of peroxiredoxin II in the mouse ovary, oviduct, uterus, and preimplantation embryo. Anat Rec (Hoboken) 293: 291–297.
[56]
Stephanou A, Latchman DS (2011) Transcriptional modulation of heat-shock protein gene expression. Biochem Res Int 2011: 238601. Available: http://www.hindawi.com/journals/bcri/201？1/238601/. Accessed 2013 Jun 11.
[57]
Arrigo AP (1998) Small stress proteins: chaperones that act as regulators of intracellular redox state and programmed cell death. Biol Chem 379: 19–26.
[58]
Swindell WR, Huebner M, Weber AP (2007) Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways. BMC Genomics 8: 125. Available: http://www.biomedcentral.com/1471-2164/8？/125. Accessed 2013 Jun 11.
[59]
Akerfelt M, Morimoto RI, Sistonen L (2010) Heat shock factors: integrators of cell stress, development and lifespan. Nat Rev Mol Cell Biol 11: 545–555.
[60]
Fink AL (1999) Chaperone-mediated protein folding. Physiol Rev 79: 425–449.
[61]
Hernebring M, Brolén G, Aguilaniu H, Semb H, Nystr？m T (2006) Elimination of damaged proteins during differentiation of embryonic stem cells. Proc Natl Acad Sci U S A 103: 7700–7705.
[62]
Akama K, Tatsuno R, Otsu M, Horikoshi T, Nakayama T, et al. (2008) Proteomic identification of differentially expressed genes in mouse neural stem cells and neurons differentiated from embryonic stem cells in vitro. Biochim Biophys Acta 1784: 773–782.
[63]
Baharvand H, Fathi A, Gourabi H, Mollamohammadi S, Salekdeh GH (2008) Identification of mouse embryonic stem cell-associated proteins. J Proteome Res 7: 412–423.
[64]
Setati MM, Prinsloo E, Longshaw VM, Murray PA, Edgar DH, et al. (2010) Leukemia inhibitory factor promotes Hsp90 association with STAT3 in mouse embryonic stem cells. IUBMB Life 62: 61–66.
[65]
Jang JH, Jung JS, Choi JI, Kang SK (2012) Nuclear Ago2/HSP60 contributes to broad spectrum of hATSCs function via Oct4 regulation. Antioxid Redox Signal 16: 383–399.
[66]
Prescott J, Coetzee GA (2006) Molecular chaperones throughout the life cycle of the androgen receptor. Cancer Lett 231: 12–19.
[67]
Odunuga OO, Longshaw VM, Blatch GL (2004) Hop: more than an Hsp70/Hsp90 adaptor protein. Bioessays 26: 1058–1068.
[68]
Longshaw VM, Baxter M, Prewitz M, Blatch GL (2009) Knockdown of the co-chaperone Hop promotes extranuclear accumulation of Stat3 in mouse embryonic stem cells. Eur J Cell Biol 88: 153–166.
[69]
Shah M, Patel K, Fried VA, Sehgal PB (2002) Interactions of STAT3 with caveolin-1 and heat shock protein 90 in plasma membrane raft and cytosolic complexes. Preservation of cytokine signaling during fever. J Biol Chem 277: 45662–45669.
[70]
Sato N, Yamamoto T, Sekine Y, Yumioka T, Junicho A, et al. (2003) Involvement of heat-shock protein 90 in the interleukin-6-mediated signaling pathway through STAT3. Biochem Biophys Res Commun 300: 847–852.
[71]
Whitesell L, Lindquist SL (2005) HSP90 and the chaperoning of cancer. Nat Rev Cancer 5: 761–772.
