Abstract
In this study, we established an in vitro model of osteogenic-inductive differentiation of rat bone marrow mesenchymal stem cells (BMSCs) to determine the mechanisms and relative gene function underlying BMSCs osteogenesis. Osteoplastic differentiation of the third generation BMSCs was induced with the α-minimal essential medium containing β-glyceraldehyde-3-phosphate, l-ascorbic acid, dexamethasone and 1,25–2(OH)2 vitamin D3 prior to applying gene chip technology (also called microarray technology) for global gene expression screening. Real-time quantitative PCR (Real-time PCR) was used to determine the temporal profile of mRNA expression of regulated genes during osteogenic differentiation of BMSCs. A bioinformatic analysis was utilized to determine the functional significance of the identified osteogenic-related genes. Purkinje cell protein 4 (Pcp4) mRNA expression was identified by the gene chip screening as being up-regulated during osteoplastic differentiation of BMSCs. Real-time PCR analysis confirmed the increased expression of Pcp4 mRNA expression during osteoplastic differentiation of BMSCs with an upward trend that peaked at day 14. The bioinformatic analysis identified Pcp4 as a gene involved in the deposition of calcium and the modulation of CaM-dependent protein kinase. Thus, we hypothesize that Pcp4 osteoplastic differentiation of BMSCs is mediated in part via Pcp4-induced calcium deposition to form mineral nodules and modulation of certain signal transduction pathways of BMPs.
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References
Caplan AI (2000) Tissue engineering designs for the future: new logics, old molecules. Tissue Eng 6:1–8
Tuan RS, Boland G, Tuli R (2003) Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther 5:32–45
Yoo JU, Johnstone B (2000) Mesenchymal stem cells and musculoskeletal repair. Cur Opin Orthop 11:391–396
Grove JE, Bruscia E, Krause DS (2004) Plasticity of bone marrow-derived stem cells. Stem Cells 22:487–500
Lin Y, Liu L, Li Z, Qiao J, Wu L, Tang W, Zheng X, Chen X, Yan Z, Tian W (2006) Pluripotency potential of human adipose-derived stem cells marked with exogenous green fluorescent protein. Mol Cell Biochem 291:1–10
Kassem M, Kristiansen M, Abdallah B (2004) Mesenchymal stem cells: cell biology and potential use in therapy. Basic Clin Pharmacol Toxicol 95:209–214
Pomerantz J, Blau HM (2004) Nuclear reprogramming: a key to stem cell function in regenerative medicine. Nat Cell Biol 6:810–816
Olsen BR, Reginato AM, Wang W (2000) Bone development. Annu Rev Cell Dev Biol 16:191–220
Harada S, Rodan GA (2003) Control of osteoblast function and regulation of bone mass. Nature 423:349–355
Wu L, Wu Y, Lin Y, Jing W, Nie X, Qiao J, Liu L, Tang W, Tian W (2007) Osteogenic differentiation of adipose derived stem cells promoted by overexpression of osterix. Mol Cell Biochem 301:83–92
Pola E, Gao W, Zhou Y, Pola R, Lattanzi W, Sfeir C, Gambotto A, Robbins PD (2004) Efficient bone formation by gene transfer of human LIM mineralization protein-3. Gene Ther 11:683–693
Yue B, Lu B, Dai KR, Zhang XL, Yu CF, Lou JR, Tang TT (2005) BMP2 gene therapy on the repair of bone defects of aged rats. Calcif Tissue Int 77:395–403
Hu Z, Peel SA, Ho SK, Sándor GK, Clokie CM (2005) Role of bovine bone morphogenetic proteins in bone matrix protein and osteoblast-related gene expression during rat bone marrow stromal cell differentiation. J Craniofac Surg 16:1006–1014
Lee KS, Kim HJ, Li QL, Chi XZ, Ueta C, Komori T, Wozney JM, Kim EG, Choi JY, Ryoo HM, Bae SC (2000) Runx2 is a common target of transforming growth factor β1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol Cell Biol 20:8783–8792
Kim HJ, Kim JH, Bae SC, Choi JY, Kim HJ, Ryoo HM (2003) The protein kinase C pathway plays a central role in the fibroblast growth factor-stimulated expression and transactivation activity of Runx2. J Biol Chem 278:319–326
Takamoto M, Tsuji K, Yamashita T, Sasaki H, Yano T, Taketani Y, Komori T, Nifuji A, Noda M (2003) Hedgehog signaling enhances core-binding factor a1 and receptor activator of nuclear factor-kappaB ligand (RANKL) gene expression in chondrocytes. J Endocrinol 177:413–421
Yamaguchi A, Komori T, Suda T (2000) Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfal. Endocr Rev 21:393–411
Bergwitz C, Prochnau A, Mayr B, Kramer FJ, Rittierodt M, Berten HL, Hausamen JE, Brabant G (2006) Identification of novel CBFA1/RUNX2 mutations causing cleidocranial dysplasia. J Inherit Metab Dis 24:648–656
Van LS, Rabie AB (2005) Mechanical strain induces Cbfa1 and type X collagen expression in mandibular condyle. Front Biosci 10:2966–2971
Ducy P, Zhang R, Geoffroy V, Ridal AL, Karsenty G (1997) Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89:747–754
Rabie AB, Tang GH, Hagg U (2004) Cbfa1 couples chondrocytes maturation and endochondral ossification in rat mandibular condylar cartilage. Arch Oral Biol 49:109–118
Kobayashi H, Gao Y, Ueta C, Yamaguchi A, Komori T (2000) Multilineage differentiation of Cbfal-deficient calvarial cells in vitro. Biochem Biophys Res Commun 273:630–636
Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B (2002) The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108:17–29
Liu L, Chen RL, Tian WD, Yan ZB, Chen XZ, Li SW, Wang T (2005) A study on the chondrogenesis of the compound of alginate gelatin and bone marrow stromal cells in vivo. Hua Xi Kou Qiang Yi Xue Za Zhi 23:60–62
International guiding principles for animal research (1985) Geneva: council for International Organization of Medical Sciences
Quackenbush J (2002) Microarray data normalization and transformation. Nat Genet 32:96–501
Krex D, Schackert HK, Schackert G (2001) Genesis of cerebral aneurysms-an update. Acta Neurochir 143:429–448
Viemann D, Goebeler M, Schmid S, Klimmek K, Sorg C, Ludwig S, Roth J (2004) Transcriptional profiling of IKK2/NF-κB– and p38 MAP kinase-dependent gene expression in TNF-α–stimulated primary human endothelial cells. Blood 103:3365–3373
Rajeevan MS, Ranamukhaarachchi DG, Vernon SD, Unger ER (2001) Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies. Methods 25:443–451
Liu W, Saint DA (2002) New quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal Biochem 302: 52–59
Simpson DA, Feeney S, Boyle C, Stitt AW (2000) Retinal VEGF mRNA measured by SYBR green I fluorescence: a versatile approach to quantitative PCR. Mol Vis 6:178–183
Shoemaker DD, Schadt EE, Armour CD, He YD, Garrett-Engele P, McDonagh PD, Loerch PM, Leonardson A, Lum PY, Cavet G, Wu LF, Altschuler SJ, Edwards S, King J, Tsang JS, Schimmack G, Schelter JM, Koch J, Ziman M, Marton MJ, Li B, Cundiff P, Ward T, Castle J, Krolewski M, Meyer MR, Mao M, Burchard J, Kidd MJ, Dai H, Phillips JW, Linsley PS, Stoughton R, Scherer S, Boguski MS (2001) Experimental annotation of the human genome using microarray technology. Nature 409: 922–927
Abe A, Inoue K, Tanaka T, Kato J, Kajiyama N, Kawaguchi R, Tanaka S, Yoshiba M, Kohara M (1999) Quantitation of hepatitis B virus genomic DNA by real-time detection PCR. J Clin Microbiol 37:2899–2903
Apple FS, Wu AH, Jaffe AS (2002) European Society of Cardiology and American College of Cardiology guidelines for redefinition of myocardial infarction: how to use existing assays clinically and for clinical trials. Am Heart J 144:981–986
Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39
Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C (2001) An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25:386–401
Girard BM, May V, Bora SH, Fina F, Braas KM (2002) Regulation of neurotrophic peptide expression in sympathetic neurons: quantitative analysis using radioimmunoassay and real-time quantitative polymerase chain reaction. Regul Pept 109:89–101
Chen H, Bouras C, Antonarakis SE (1996) Cloning of the cDNA for a human homolog of the rat PEP-19 gene and mapping to chromosome 21q22.2–q22.3. Hum Genet 98:672–677
Sala A, Scaturro M, Proia P, Schiera G, Balistreri E, Aflalo-Rattenbach R, Créau N, Di Liegro I (2007) Cloning of a rat-specific long PCP4/PEP19 isoform. Int J Mol Med 19:501–509
Thomas S, Thiery E, Aflalo R, Vayssettes C, Verney C, Berthuy I, Creau N (2003) PCP4 is highly expressed in ectoderm and particularly in neuroectoderm derivatives during mouse embryogenesis. Gene Expr Patterns 3:93–97
Li MZ, Wang JS, Jiang DJ, Xiang CX, Wang FY, Zhang KH, Williams PR, Chen ZF (2006) Molecular mapping of developing dorsal horn-enriched genes by microarray and dorsal/ventral subtractive screening. Dev Biol 292:555–564
Murray KD, Choudary PV, Jones EG (2007) Nucleus- and cell-specific gene expression in monkey thalamus. Proc Natl Acad Sci USA 104:1989–1994
Iwamoto K, Bundo M, Yamamoto M, Ozawa H, Saito T, Kato T (2004) Decreased expression of NEFH and PCP4/PEP19 in the prefrontal cortex of alcoholics. Neurosci Res 49:379–385
Dickerson JB, Morgan MA, Mishra A, Slaughter CA, Morgan JI, Zheng J (2006) The influence of phosphorylation on the activity and structure of the neuronal IQ motif protein, PEP-19. Brain Res 1092:16–27
Slemmon JR, Feng B, Erhardt JA (2000) Small proteins that modulate calmodulin-dependent signal transduction: effects of PEP-19, neuromodulin, and neurogranin on enzyme activation and cellular homeostasis. Mol Neurobiol 22:99–113
Putkey JA, Kleerekoper Q, Gaertner TR, Waxham MN (2003) A new role for IQ motif proteins in regulating calmodulin function. J Biol Chem 278:49667–49670
Acknowledgments
We thank Miss Xiaoyu Li for excellent technical support. This work was supported by the Chinese National Natural Science Foundation (30100210, 30572051) and the Outstanding Young Academic Leaders Foundation of Sichuan Province (06ZQ026–008).
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Xiao, J., Wu, Y., Chen, R. et al. Expression of Pcp4 gene during osteogenic differentiation of bone marrow mesenchymal stem cells in vitro. Mol Cell Biochem 309, 143–150 (2008). https://doi.org/10.1007/s11010-007-9652-x
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DOI: https://doi.org/10.1007/s11010-007-9652-x