Constitutive expression of thrombospondin 1 in MC3T3-E1 osteoblastic cells inhibits mineralization

Authors

  • Akemichi Ueno,

    1. Department of Molecular Biology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
    Current affiliation:
    1. Department of Hygiene Chemistry, School of Pharmaceutical Sciences, Ohu University, Koriyama, Fukushima 963-8611, Japan.
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  • Yoshihiro Miwa,

    1. Department of Biochemistry, School of Dentistry, The University of Tokushima, Tokushima, Japan
    Current affiliation:
    1. Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
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  • Keiko Miyoshi,

    1. Department of Molecular Biology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
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  • Taigo Horiguchi,

    1. Department of Molecular Biology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
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  • Hideo Inoue,

    1. Department of Biochemistry, School of Dentistry, The University of Tokushima, Tokushima, Japan
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  • Intan Ruspita,

    1. Department of Molecular Biology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
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  • Kaori Abe,

    1. Department of Molecular Biology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
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  • Kikuji Yamashita,

    1. Department of Anatomy, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
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  • Eiji Hayashi,

    1. Department of Oral and Maxillofacial Surgery I, School of Dentistry, The University of Tokushima, Tokushima, Japan
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  • Takafumi Noma

    Corresponding author
    1. Department of Molecular Biology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
    • Department of Molecular Biology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan.
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Abstract

Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3-E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18- and 24-mer antisense oligonucleotides caused concentration-dependent increases in the number of mineralized nodules, acid-soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide-treated MC3T3-E1 cells, thickened extracellular matrix, well-developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3-E1 cells. Furthermore, MC3T3-E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose-dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis. J. Cell. Physiol. 209: 322–332, 2006. © 2006 Wiley-Liss, Inc.

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