Comprehensive Microarray Analysis of Bone Morphogenetic Protein 2‐Induced Osteoblast Differentiation Resulting in the Identification of Novel Markers for Bone Development

Osteoblasts are cells responsible for matrix deposition during bone development and although temporal expression of many genes has been related to osteoblast differentiation, a complete description of osteoblast‐specific gene regulation will lead to a better understanding of osteoblast function. In this study, microarray technology was used to analyze gene expression on a broad scale during osteoblast differentiation. Expression analysis of 9596 sequences revealed 342 genes and expressed sequence tags (ESTs) to be modulated differentially during a time course experiment in which murine C2C12 mesenchymal progenitor cells were induced to differentiate into mature osteoblasts by treatment with bone morphogenetic protein 2 (BMP‐2). By means of hierarchical clustering, these genes were grouped by similarities in their expression profiles, resulting in subsets of early, intermediate, and late response genes, which are representative of the distinct stages of osteoblast differentiation. To identify new bone markers, the bone specificity of the late response genes was determined by comparing BMP‐induced expression in C2C12 and MC3T3 osteoblasts with that in NIH3T3 fibroblasts. This resulted in the identification of nine novel genes and ESTs that were induced specifically in osteoblasts, in addition to the well‐known markers ALP and osteocalcin. For at least one of these novel genes, Wnt inhibitory factor 1, and two of the ESTs, expression in developing bone was verified in vivo by in situ hybridization of E16.5 mouse embryos. In conclusion, by a combination of in vitro and in vivo screening approaches, a set of new genes related to osteoblast differentiation and skeletal development has been identified.

[1]  Stuart A. Aaronson,et al.  Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow , 2001, Nature Cell Biology.

[2]  G. Stein,et al.  Transcriptional control of osteoblast differentiation. , 1998, Biochemical Society transactions.

[3]  A. Seth,et al.  Coordinate Expression of Novel Genes During Osteoblast Differentiation , 2000, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[4]  J. Nathans,et al.  A new secreted protein that binds to Wnt proteins and inhibits their activites , 1999, Nature.

[5]  A. Hoffmann,et al.  Bmp‐2 downstream targets in mesenchymal development identified by subtractive cloning from recombinant mesenchymal progenitors (C3H10T½) , 1998, Developmental dynamics : an official publication of the American Association of Anatomists.

[6]  G. Karsenty,et al.  Osf2/Cbfa1: A Transcriptional Activator of Osteoblast Differentiation , 1997, Cell.

[7]  G. Stein,et al.  Structure of the rat osteocalcin gene and regulation of vitamin D-dependent expression. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[8]  S. Mohan,et al.  Insulin‐like Growth Factor (IGF)‐I, ‐II, IGF Binding Proteins (IGFBP)−3, −4, and −5 Levels in the Conditioned Media of Normal Human Bone Cells Are Skeletal Site‐Dependent , 1997, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[9]  R. Behringer,et al.  Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein , 1997, Nature.

[10]  Y. Koshihara,et al.  Involvement of type VI collagen in interleukin-4-induced mineralization by human osteoblast-like cells in vitro. , 1999, Biochimica et biophysica acta.

[11]  F. Lecanda,et al.  Relative abundance of different cadherins defines differentiation of mesenchymal precursors into osteogenic, myogenic, or adipogenic pathways , 2000, Journal of cellular biochemistry.

[12]  I. Thesleff,et al.  Shh, Bmp-2, Bmp-4 and Fgf-8 are associated with initiation and patterning of mouse tongue papillae , 1999, Mechanisms of Development.

[13]  A. Seth,et al.  Discovery of osteoblast-associated genes using cDNA microarrays. , 2002, Bone.

[14]  Ronald W. Davis,et al.  Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray , 1995, Science.

[15]  R. Iozzo.  The Biology of the Small Leucine-rich Proteoglycans , 1999, The Journal of Biological Chemistry.

[16]  M. Somerman,et al.  Matrix γ-Carboxyglutamic Acid Protein Is a Key Regulator of PTH-Mediated Inhibition of Mineralization in MC3T3-E1 Osteoblast-Like Cells. , 2001, Endocrinology.

[17]  Matthew H. Kaufman,et al.  The Atlas of Mouse Development , 1992 .

[18]  K. Miyata,et al.  Interleukin-11 induces osteoblast differentiation and acts synergistically with bone morphogenetic protein-2 in C3H10T1/2 cells. , 2001, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[19]  Yusuke Nakamura,et al.  Genome-wide screening by cDNA microarray of genes associated with matrix mineralization by human mesenchymal stem cells in vitro. , 2002, Biochemical and biophysical research communications.

[20]  J. Deng,et al.  The Novel Zinc Finger-Containing Transcription Factor Osterix Is Required for Osteoblast Differentiation and Bone Formation , 2002, Cell.

[21]  G. Stein,et al.  Differential regulation of the two principal Runx2/Cbfa1 n-terminal isoforms in response to bone morphogenetic protein-2 during development of the osteoblast phenotype. , 2001, Endocrinology.

[22]  Sakae Tanaka,et al.  Negative Regulation of BMP/Smad Signaling by Tob in Osteoblasts , 2000, Cell.

[23]  D. Botstein,et al.  Cluster analysis and display of genome-wide expression patterns. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[24]  B. L. Riggs,et al.  Assessment of Gene Regulation by Bone Morphogenetic Protein 2 in Human Marrow Stromal Cells Using Gene Array Technology , 2001, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[25]  G. Beck,et al.  Gene array analysis of osteoblast differentiation. , 2001, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[26]  A. Boskey,et al.  Effects of Bone CS-Proteoglycans, DS-Decorin, and DS-Biglycan on Hydroxyapatite Formation in a Gelatin Gel , 1997, Calcified Tissue International.

[27]  V. Rosen,et al.  Differential Roles for Bone Morphogenetic Protein (BMP) Receptor Type IB and IA in Differentiation and Specification of Mesenchymal Precursor Cells to Osteoblast and Adipocyte Lineages , 1998, The Journal of cell biology.

[28]  T. Yoneda,et al.  Patterns of gene expression associated with BMP-2-induced osteoblast and adipocyte differentiation of mesenchymal progenitor cell 3T3-F442A , 2000, Journal of Bone and Mineral Metabolism.

[29]  G. Rodan,et al.  Structure and expression of rat osteosarcoma (ROS 17/2.8) alkaline phosphatase: product of a single copy gene. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[30]  B. Hogan,et al.  Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A). , 1990, Development.

[31]  Roland Somogyi,et al.  Large Scale Gene Expression Analysis of Cholesterol-loaded Macrophages* , 2000, The Journal of Biological Chemistry.

[32]  S. Keränen,et al.  The life history of an embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. , 1998, Development.

[33]  L. Wodicka,et al.  Genome-wide expression monitoring in Saccharomyces cerevisiae , 1997, Nature Biotechnology.

[34]  Makoto Sato,et al.  Targeted Disruption of Cbfa1 Results in a Complete Lack of Bone Formation owing to Maturational Arrest of Osteoblasts , 1997, Cell.

[35]  T. Fournier,et al.  Alpha-1-acid glycoprotein. , 2000, Biochimica et biophysica acta.

[36]  M. Goumans,et al.  Expression of activin subunits, activin receptors and follistatin in postimplantation mouse embryos suggests specific developmental functions for different activins. , 1994, Development.

[37]  Miikka Vikkula,et al.  LDL Receptor-Related Protein 5 (LRP5) Affects Bone Accrual and Eye Development , 2001, Cell.

[38]  G. Stein,et al.  Transient upregulation of CBFA1 in response to bone morphogenetic protein‐2 and transforming growth factor β1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation , 1999, Journal of cellular biochemistry.

[39]  G. Karsenty,et al.  The osteoblast: a sophisticated fibroblast under central surveillance. , 2000, Science.

[40]  H. Frierson,et al.  Osteocalcin is incompletely spliced in non-osseous tissues. , 2001, Gene.

[41]  E. Mackie,et al.  Modulation of osteoblast behaviour by tenascin. , 1996, Journal of cell science.