Expression and regulation of Ly-6 differentiation antigens by murine osteoblasts.

Abstract

Osteoblasts arise from mesenchymal stem cells and differentiate to become osteoid-secreting cells. However, little is known about these cells during their stages of differentiation. One reason for this lack of information is that there is no reliable method to identify osteoblasts as they mature. One method that has been used successfully with other cell types is the identification of plasma membrane-expressed differentiation antigens. The Ly-6 multigene family encodes differentiation antigens originally detected on lymphoid cells. Primary murine osteoblasts and the osteoblast-like MC3T3 cell line were examined for expression of Ly-6 antigens by flow cytometry. Primary osteoblasts and MC3T3 cells constitutively expressed both Ly-6A and Ly-6C antigens, although Ly-6C was much less abundant. Antigen expression was markedly increased by pretreating the cells with interferon-alpha/beta or -gamma. Northern blot analysis revealed constitutively expression of Ly-6 messenger RNA that was up-regulated by interferon treatment. Pretreatment of the cells with transforming growth factor-beta 1 or 1,25-dihydroxyvitamin D3 diminished constitutive Ly-6 expression. Ly-6 was localized intracellularly to the Golgi complex. The current results demonstrate that mature osteoblasts express on their cell surface specific Ly-6 antigens in a pattern that distinguishes them from the surrounding bone marrow cells. These studies represent the first identification of osteoblast differentiation antigens that can be directly related to cells within the hematopoietic lineage. By identifying similar antigens, osteoblasts at various stages of differentiation may be identified, isolated, and characterized.

Megakaryocytes Regulate Expression of Pyk2 Isoforms and Caspase-mediated Cleavage of Actin in Osteoblasts

“…Preparation of Neonatal Calvarial Osteoblasts-Murine calvarial cells were prepared using a modification of a protocol described previously (15)(16)(17)(18). Briefly, calvaria from 2-to 3-day neonatal mice were pretreated with 10 mM EDTA in phosphate-buffered saline (PBS) for 30 min.…”

“…The calvaria were then subject to sequential collagenase digestions, and cells were collected. Fractions 3-5 were used as the starting population that consisted of ϳ95% OB or OB precursors, as determined by a variety of criteria (15,17,18). For experiments with MKs, OBs were seeded at 2 ϫ 10 4 cells/ml in 6-well plates or in 10-cm 2 dishes.…”

Pax5-Deficient Mice Exhibit Early Onset Osteopenia with Increased Osteoclast Progenitors

“…Murine calvarial OB were prepared as previously described (12). In brief, calvaria from mice Ͻ48 h old were pretreated with EDTA in PBS for 30 min and then subjected to sequential collagenase digestion (Worthington Biomedical, Lakewood, NJ), and cells collected from fractions 3-5 were used as the starting population.…”

Impact of interactions of cellular components of the bone marrow microenvironment on hematopoietic stem and progenitor cell function

“…Calvarial OBs were prepared after a modification of published methods. 23,24 Calvariae from C57BL/6 mice less than 48 hours old were dissected, pretreated with ethylenediaminetetraacetic acid in phosphate-buffered saline (PBS) for 30 minutes, and then subjected to sequential collagenase digestions (200 U/mL). Fractions 3 to 5 (collected between 45-60, 60-75, and 75-90 minutes through the digestion) were collected and used as OBs.…”

“…These cells are more than 95% OBs or OB precursors as previously demonstrated. 24 Freshly prepared OBs were used for all studies.Two-day long bone OBs. Neonatal long bones (tibiae and femurs) were dissected from C57BL/6 mice less than 48 hours old.…”