Immortalization and characterization of osteoblast cell lines generated from wild-type and Nmp4-null mouse bone marrow stromal cells using murine telomerase reverse transcriptase (mTERT)

Authors

  • Marta B. Alvarez,

    1. Department of Anatomy & Cell Biology, Indiana University School of Medicine (IUSM), Indianapolis, Indiana
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  • Paul Childress,

    1. Department of Anatomy & Cell Biology, Indiana University School of Medicine (IUSM), Indianapolis, Indiana
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  • Binu K. Philip,

    1. Drug Safety and Evaluation, Bristol-Myers Squibb, Mt. Vernon, Indiana
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  • Rita Gerard-O'Riley,

    1. Department of Cellular and Integrative Physiology, Indiana University School of Medicine (IUSM), Indianapolis, Indiana
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  • Michael Hanlon,

    1. Department of Anatomy & Cell Biology, Indiana University School of Medicine (IUSM), Indianapolis, Indiana
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  • Brittney-Shea Herbert,

    1. Department of Medical and Molecular Genetics, Indiana University School of Medicine (IUSM), Indianapolis, Indiana
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  • Alexander G. Robling,

    1. Department of Anatomy & Cell Biology, Indiana University School of Medicine (IUSM), Indianapolis, Indiana
    2. Department of Biomedical Engineering, Indiana University-Purdue University at Indianapolis, Indiana
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  • Fredrick M. Pavalko,

    Corresponding author
    1. Department of Cellular and Integrative Physiology, Indiana University School of Medicine (IUSM), Indianapolis, Indiana
    • Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Medical Science Bldg 5035, 635 Barnhill Drive, Indianapolis, IN 46202.
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  • Joseph P. Bidwell

    Corresponding author
    1. Department of Anatomy & Cell Biology, Indiana University School of Medicine (IUSM), Indianapolis, Indiana
    • Department of Anatomy & Cell Biology, Indiana University School of Medicine, Medical Science Bldg 5035, 635 Barnhill Drive, Indianapolis, IN 46202.
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  • Conflict of interest: None.

Abstract

Intermittent parathyroid hormone (PTH) adds new bone to the osteoporotic skeleton; the transcription factor Nmp4/CIZ represses PTH-induced bone formation in mice and as a consequence is a potential drug target for improving hormone clinical efficacy. To explore the impact of Nmp4/CIZ on osteoblast phenotype, we immortalized bone marrow stromal cells from wildtype (WT) and Nmp4-knockout (KO) mice using murine telomerase reverse transcriptase. Clonal lines were initially chosen based on their positive staining for alkaline phosphatase and capacity for mineralization. Disabling Nmp4/CIZ had no gross impact on osteoblast phenotype development. WT and KO clones exhibited identical sustained growth, reduced population doubling times, extended maintenance of the mature osteoblast phenotype, and competency for differentiating toward the osteoblast and adipocyte lineages. Additional screening of the immortalized cells for PTH-responsiveness permitted further studies with single WT and KO clones. We recently demonstrated that PTH-induced c-fos femoral mRNA expression is enhanced in Nmp4-KO mice and in the present study we observed that hormone stimulated either an equivalent or modestly enhanced increase in c-fos mRNA expression in both primary null and KO clone cells depending on PTH concentration. The null primary osteoblasts and KO clone cells exhibited a transiently enhanced response to bone morphogenetic protein 2 (BMP2). The clones exhibited lower and higher expressions of the PTH receptor (Pthr1) and the BMP2 receptor (Bmpr1a, Alk3), respectively, as compared to primary cells. These immortalized cell lines will provide a valuable tool for disentangling the complex functional roles underlying Nmp4/CIZ regulation of bone anabolism. J. Cell. Physiol. 227: 1873–1882, 2012. © 2011 Wiley Periodicals, Inc.

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