Conflict of interest: None.
Original Research Article
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)†
Article first published online: 23 JAN 2012
Copyright © 2011 Wiley Periodicals, Inc.
Journal of Cellular Physiology
Volume 227, Issue 5, pages 1873–1882, May 2012
How to Cite
Alvarez, M. B., Childress, P., Philip, B. K., Gerard-O'Riley, R., Hanlon, M., Herbert, B.-S., Robling, A. G., Pavalko, F. M. and Bidwell, J. P. (2012), 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). J. Cell. Physiol., 227: 1873–1882. doi: 10.1002/jcp.22915
- Issue published online: 23 JAN 2012
- Article first published online: 23 JAN 2012
- Accepted manuscript online: 5 JUL 2011 10:28AM EST
- Manuscript Accepted: 21 JUN 2011
- Manuscript Received: 17 JUN 2011
- National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Grant Number: DK053796
- The National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS). Grant Number: AR052682
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.