Abstract
Human mesenchymal stem cells (hMSC) are a population of multipotent cells that can differentiate into osteoblasts, chondrocytes, adipocytes, and other cells. The exact mechanism governing the differentiation of hMSC into osteoblasts remains largely unknown. Here, we analyzed protein expression profiles of undifferentiated as well as osteogenic induced hMSC using 2-D gel electrophoresis (2-DE), mass spectrometry (MS), and peptide mass fingerprinting (PMF) to investigate the early gene expression in osteoblast differentiation. We have generated proteome maps of undifferentiated hMSC and osteogenic induced hMSC on day 3 and day 7. 2-DE revealed 102 spots with at least 2.0-fold changes in expression and 52 differently expressed proteins were successfully identified by MALDI-TOF-MS. These proteins were classified into 7 functional categories: metabolism, signal transduction, transcription, calcium-binding protein, protein degradation, protein folding and others. The expression of some identified proteins was confirmed by further RT-PCR analyses. This study clarifies the global proteome during osteoblast differentiation. Our results will play an important role in better elucidating the underlying molecular mechanism in hMSC differentiation into osteoblasts.
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Abbreviations
- HMSC:
-
Human mesenchymal stem cells
- MW:
-
Molecular weight
References
Marie PJ (2003) Fibroblast growth factor signaling controlling osteoblast differentiation. Gene 316:23–32
Pittenger MF, Mackay AM, Beck SC et al (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147
Hishikawa K, Miura S, Marumo T et al (2004) Gene expression profile of human mesenchymal stem cells during osteogenesis in three-dimensional thermoreversible gelation polymer. Biochem Biophys Res Commun 317:1103–1107
Yamaguchi A, Komori T, Suda T (2000) Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr Rev 21:393–411
Doi M, Nagano A, Nakamura Y (2002) Genome-wide screening by cDNA microarray of genes associated with matrix mineralization by human mesenchymal stem cells in vitro. Biochem Biophys Res Commun 290:381–390
Lee RH, Kim B, Choi I et al (2004) Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem 14:311–324
Zenzmaier C, Gesslbauer B, Grobuschek N et al (2005) Proteomic profiling of human stem cells derived from umbilical cord blood. Biochem Biophys Res Commun 328:968–972
Yang JW, Rodrigo R, Felipo V et al (2005) Proteome analysis of primary neurons and astrocytes from rat cerebellum. J Proteome Res 4:768–788
Heim M, Frank O, Kampmann G et al (2004) The phytoestrogen genistein enhances osteogenesis and represses adipogenic differentiation of human primary bone marrow stromal cells. Endocrinology 145:848–859
Jaiswal N, Haynesworth SE, Caplan AI et al (1997) Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 64:295–312
Cheng SL, Yang JW, Rifas L et al (1994) Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology 134:277–286
Foster LJ, Zeemann PA, Li C et al (2005) Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation. Stem Cells 23:1367–1377
Salasznyk RM., Westcott AM, Klees RF et al (2005) Comparing the protein expression profiles of human mesenchymal stem cells and human osteoblasts using gene ontologies. Stem Cells Dev 14:354–366
Digirolamo CM, Stokes D, Colter D et al (1999) Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol 107:275–281
Grigoriadis AE, Heersche JN, Aubin JE (1988) Differentiation of muscle, fat, cartilage, and bone from progenitor cells present in a bone-derived clonal cell population: effect of dexamethasone. J Cell Biol 106:2139–2151
Stenderup K, Justesen J, Eriksen EF et al (2001) Number and proliferative capacity of osteogenic stem cells are maintained during aging and in patients with osteoporosis. J Bone Miner Res 16:1120–1129
Loeffler-Ragg J, Skvortsov S, Sarg B et al (2005) Gefitinib-responsive EGFR-positive colorectal cancers have different proteome profiles from non-responsive cell lines. Eur J Cancer 41:2338–2346
Maurer MH, Feldmann RE Jr et al (2003) The proteome of neural stem cells from adult rat hippocampus. Proteome Sci 1:4
Gorg A, Obermaier C, Boguth G et al (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21:1037–1053
Prowse AB, McQuade LR, Bryant KJ et al (2005) A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells. Proteomics 5:978–989
Miura Y, Kano M, Abe K et al (2005) Age-dependent variations of cell response to oxidative stress: proteomic approach to protein expression and phosphorylation. Electrophoresis 26:2786–2796
Hiratsuka M, Inoue T, Toda T et al (2003) Proteomics-based identification of differentially expressed genes in human gliomas: down-regulation of SIRT2 gene. Biochem Biophys Res Commun 309:558–566
Nishigaki R, Osaki M, Hiratsuka M et al (2005) Proteomic identification of differentially-expressed genes in human gastric carcinomas. Proteomics 5:3205–3213
Kirsch T, Nah HD, Shapiro IM et al (1997) Regulated production of mineralization-competent matrix vesicles in hypertrophic chondrocytes. J Cell Biol 137:1149–1160
Genge BR, Wu LN, Wuthier RE (1989) Identification of phospholipid-dependent calcium-binding proteins as constituents of matrix vesicles. J Biol Chem 264:10917–10921
Wang W, Kirsch T (2002) Retinoic acid stimulates annexin-mediated growth plate chondrocyte mineralization. J Cell Biol 157:1061–1069
Gillette JM, Nielsen-Preiss SM (2004) The role of annexin 2 in osteoblastic mineralization. J Cell Sci 117:441–449
Kirsch T, Harrison G, Golub EE et al (2000) The roles of annexins and types II and X collagen in matrix vesicle-mediated mineralization of growth plate cartilage. J Biol Chem 275:35577–35583
Haut Donahue TL, Genetos DC, Jacobs CR et al (2004) Annexin V disruption impairs mechanically induced calcium signaling in osteoblastic cells. Bone 35:656–663
William F, Mroczkowski B, Cohen S et al (1988) Differentiation of HL-60 cells is associated with an increase in the35-kDa protein lipocortin I. J Cell Physiol 137:402–410
Ye NS, Chen J, Luo GA et al (2006) Proteomic profiling of rat bone marrow mesenchymal stem cells induced by 5-azacytidine. Stem Cells Dev 15:665–676
Ghosh-Choudhury N, Abboud SL, Nishimura R et al (2002) Requirement of BMP-2-induced phosphatidylinositol 3-kinase and Akt serine/threonine kinase in osteoblast differentiation and Smad-dependent BMP-2 gene transcription. J Biol Chem 277:33361–33368
Osyczka AM, Leboy PS (2005) Bone morphogenetic protein regulation of early osteoblast genes in human marrow stromal cells is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling. Endocrinology 146:3428–3437
Shoba LN, Lee JC (2003) Inhibition of phosphatidylinositol 3-kinase and p70S6 kinase blocks osteogenic protein-1 induction of alkaline phosphatase activity in fetal rat calvaria cells. J Cell Biochem 88:1247–1255
Fujita T, Azuma Y, Fukuyama R et al (2004) Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling. J Cell Biol 166:85–95
Lehto M, Tienari J, Lehtonen S et al (2004) Subfamily III of mammalian oxysterol-binding protein (OSBP) homologues: the expression and intracellular localization of ORP3, ORP6, and ORP7. Cell Tissue Res 315:39–57
Lehto M, Laitinen S, Chinetti G et al (2001) The OSBP-related protein family in humans. J Lipid Res 42:1203–1213
Lehto M, Olkkonen VM (2003) The OSBP-related proteins: a novel protein family involved in vesicle transport, cellular lipid metabolism, and cell signalling. Biochim Biophys Acta 1631:1–11
Im YJ, Raychaudhuri S, Prinz WA et al (2005) Structural mechanism for sterol sensing and transport by OSBP-related proteins. Nature 437:154–158
Wang PY, Weng J, Anderson RG (2005) OSBP is a cholesterol-regulated scaffolding protein in control of ERK 1/2 activation. Science 307:1472–1476
Jaiswal RK, Jaiswal N, Bruder SP et al (2000) Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J Biol Chem 275:9645–9652
Klees RF, Salasznyk RM, Kingsley K et al (2005) Laminin-5 induces osteogenic gene expression in human mesenchymal stem cells through an ERK-dependent pathway. Mol Biol Cell 16:881–890
Hubler TR, Denny WB, Valentine DL et al (2003) The FK506-binding immunophilin FKBP51 is transcriptionally regulated by progestin and attenuates progestin responsiveness. Endocrinology 144:2380–2387
Patterson CE, Gao J, Rooney AP et al (2002) Genomic organization of mouse and human 65 kDa FK506-binding protein genes and evolution of the FKBP multigene family. Genomics 79:881–889
Bouwmeester T, Bauch A, Ruffner H et al (2004) A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway. Nat Cell Biol 6:97–105
Moreau A, Yotov WV, Glorieux FH et al (1998) Bone-specific expression of the alpha chain of the nascent polypeptide-associated complex, a coactivator potentiating c-Jun-mediated transcription. Mol Cell Biol 18:1312–1321
Merriman HL, La Tour D, Linkhart TA et al (1990) Insulin-like growth factor-I and insulin-like growth factor-II induce c-fos in mouse osteoblastic cells. Calcif Tissue Int 46:258–262
McCabe LR, Kockx M, Lian J et al (1995) Selective expression of fos- and jun-related genes during osteoblast proliferation and differentiation. Exp Cell Res 218:255–262
Candeliere GA, Prud’homme J, St Arnaud R (1991) Differential stimulation of fos and jun family members by calcitriol in osteoblastic cells. Mol Endocrinol 5:1780–1788
Yotov WV, Moreau A, St Arnaud R (1998) The alpha chain of the nascent polypeptide-associated complex functions as a transcriptional coactivator. Mol Cell Biol 18:1303–1311
Stilo R, Liguoro D, di Jeso B et al (2003) The alpha-chain of the nascent polypeptide-associated complex binds to and regulates FADD function. Biochem Biophys Res Commun 303:1034–1041
Cohen GM (1997) Caspases: the executioners of apoptosis. Biochem J 326:1–16
Kischkel FC, Hellbardt S, Behrmann I et al (1995) Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J 14:5579–5588
Mogi M, Togari A (2003) Activation of caspases is required for osteoblastic differentiation. J Biol Chem 278:47477–47482
Monroe DG, Secreto FJ, Subramaniam M et al (2005) Estrogen receptor alpha and beta heterodimers exert unique effects on estrogen- and tamoxifen-dependent gene expression in human U2OS osteosarcoma cells. Mol Endocrinol 19:1555–68
Monroe DG, Secreto FJ, Hawse JR et al (2006) Estrogen receptor isoform-specific regulation of the retinoblastoma-binding protein 1 (RBBP1) gene: roles of AF1 and enhancer elements. J Biol Chem 281:28596–604
Russell RG, Lasorella A, Dettin LE et al (2004) Id2 drives differentiation and suppresses tumor formation in the intestinal epithelium. Cancer Res 64:7220–5
Beyea JA, Olson DM, Harvey S (2005) Growth hormone (GH) action in the developing lung: changes in lung proteins after adenoviral GH overexpression. Dev Dyn 234:404–12
Vuadens F, Gasparini D, Deon C et al (2002) Identification of specific proteins in different lymphocyte populations by proteomic tools. Proteomics 2:105–11
Vuadens F, Rufer N, Kress A et al (2004) Identification of swiprosin 1 in human lymphocytes. Proteomics 4:2216–2220
Kawano Y, Yoshimura T, Tsuboi D et al (2005) CRMP-2 is involved in kinesin-1-dependent transport of the Sra-1/WAVE1 complex and axon formation. Mol Cell Biol 25:9920–35
Inagaki N, Chihara K, Arimura N et al (2001) CRMP-2 induces axons in cultured hippocampal neurons. Nat Neurosci 4:781–2
Kitamura K, Takayama M, Hamajima N et al (1999) Characterization of the human dihydropyrimidinase-related protein 2 (DRP-2) gene. DNA Res 6:291–7
Ulloa L, Batliwalla FM, Andersson U et al (2003) High mobility group box chromosomal protein 1 as a nuclear protein, cytokine, and potential therapeutic target in arthritis. Arthritis Rheum 48:876–81
Palumbo R, Bianchi ME (2004) High mobility group box 1 protein, a cue for stem cell recruitment . Biochem Pharmacol 68:1165–1170
Merenmies J, Pihlaskari R, Laitinen J et al (1991) 30-kDa heparin-binding protein of brain (amphoterin) involved in neurite outgrowth: amino acid sequence and localization in the filopodia of the advancing plasma membrane. J Biol Chem 266:16722–9
Taguchi A, Blood DC, del Toro G et al (2000) Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases. Nature 405:354–60
Edgar AJ, Dover SL, Lodrick MN et al (2005) Bone morphogenetic protein-2 induces expression of murine zinc finger transcription factor ZNF450. J Cell Biochem 94:202–215
Qi H, Aguiar DJ, Williams SM et al (2003) Identification of genes responsible for osteoblast differentiation from human mesodermal progenitor cells. Proc Natl Acad Sci U S A 100:3305–3310
Nakashima K, Zhou X, Kunkel G et al (2002) The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108:17–29
Celil AB, Campbell PG (2005) BMP-2 and insulin-like growth factor-I mediate Osterix (Osx) expression in human mesenchymal stem cells via the MAPK and protein kinase D signaling pathways. J Biol Chem 280:31353–31359
Acknowledgments
This work was supported by the National Natural Science Foundation of China (30100188, 30571891), National Basic Research Program of China (2001CB509904), the Key Scientific and Technological Projects of Guangdong Province (2003A3020103, 2005A30201001), and the Key Scientific and Technological Projects of Guangzhou City, No. 2002U13E0011. We thank all colleagues of Center for Stem Cell Biology and Tissue Engineering of Sun Yat-sen University in China for the technical support.
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Fig. S1 The expressions of 7 protein spots on the 2-DE afterosteoblast differentiation. 0 d: undifferentiated hMSC. 3 d, 7 d: days after treating inducing media (PDF 84 kb)
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Zhang, AX., Yu, WH., Ma, BF. et al. Proteomic identification of differently expressed proteins responsible for osteoblast differentiation from human mesenchymal stem cells. Mol Cell Biochem 304, 167–179 (2007). https://doi.org/10.1007/s11010-007-9497-3
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DOI: https://doi.org/10.1007/s11010-007-9497-3