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)

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.

Nmp4/CIZ Suppresses the Parathyroid Hormone Anabolic Window by Restricting Mesenchymal Stem Cell and Osteoprogenitor Frequency

“…This is consistent with the observed equivalent addition of bone during the first 2 weeks of treatment, but the divergence in both serum osteocalcin and bone formation in the null mice at 3 weeks [7 and the present work]. Finally, the Nmp4/CIZ-KO osteoblast exhibits a modest, but significant, enhanced response to numerous anabolic stimuli, including PTH, BMP2, and mechanical loading [24,[33][34][35]; therefore, an expanded population of such cells is certainly consistent with the augmented skeletal bone mineral density and bone mineral content of the null animals.…”

“…To address this issue, a combination of genetic-, drug-, and transplantation-based approaches will be required, because all of these methods have strengths and drawbacks, yet their intersection reveals complementary aspects of the phenomenon under study. However, the previously observed heightened PTH-responsiveness and osteogenic capacity of Nmp4-KO BMSCs and osteoblasts in culture [24,33,35] and the enhanced number of the progenitors of these cells (present study) likely make a substantial contribution to the extended anabolic window. Additionally, Nmp4/CIZ deficiency augmented newly formed trabecular bone mass after femoral BM ablation as compared to WT mice [24], confirming the enhanced osteogenic capacity of the reconstituted KO BM.…”

Nmp4/CIZ Closes the Parathyroid Hormone Anabolic Window

“…65,67 As previously indicated, PTH-induced c-fos mRNA expression was transiently enhanced in Nmp4 -KO femoral bone, compared to WT bone, one hour after the last hormone injection at two weeks of PTH treatment. 25 Consistent with the in vivo observation, primary Nmp4 -KO BMSC-derived osteoblasts and an Nmp4 -null clonal cell line both showed a modest concentration-dependent enhancement in PTH-stimulated increases in c-fos mRNA expression as compared to that observed in WT bone cells.…”

“…26 Finally, we did not discern any differences in these parameters between WT and null immortalized clonal cell lines. 67 …”

“…25 Consistent with the in vivo observation, primary Nmp4 -KO BMSC-derived osteoblasts and an Nmp4 -null clonal cell line both showed a modest concentration-dependent enhancement in PTH-stimulated increases in c-fos mRNA expression as compared to that observed in WT bone cells. 67 …”