Gori, Francesca

Abstract Osteoporosis is a serious complication of systemic glucocorticoid use. However, while glucocorticoids increase bone resorption in vitro and in vivo, the mechanism(s) of this effect are at present unclear. Recent studies have identified the osteoprotegerin (OPG) ligand (OPG-L) as the final effector of osteoclastogenesis, an action that is opposed by the soluble neutralizing receptor, OPG. Thus, we assessed glucocorticoid regulation of OPG and OPG-L in various human osteoblastic lineage cells using Northern analysis, RT-PCR, and ELISA. Dexamethasone inhibited constitutive OPG messenger RNA (mRNA) steady-state levels by 70–90% in primary (MS) and immortalized stromal cells (hMS), primary trabecular osteoblasts (hOB), immortalized fetal osteoblasts (hFOB), and osteosarcoma cells (MG-63). In hFOB cells, dexamethasone inhibited constitutive OPG mRNA steady-state levels in a dose- and time-dependent fashion by 90%, and also suppressed cytokine-stimulated OPG mRNA steady-state levels. Dexamethasone-induced inhibition of OPG mRNA levels was not affected by the protein synthesis inhibitor, cycloheximide, and was shown to be due to inhibition of OPG gene transcription using a nuclear run-on assay. Moreover, dexamethasone also dose dependently (10−10m–10−7m) inhibited constitutive OPG protein concentrations in the conditioned medium of hFOB cells from 2.59 ± 0.02 ng/ml (control) to 0.30 ± 0.01 ng/ml (88% inhibition; P < 0.001 by ANOVA). Concurrently, dexamethasone stimulated OPG-L mRNA steady-state levels in MS and hFOB cells by 2- and 4-fold, respectively. Treatment of murine marrow cultures with conditioned medium harvested from dexamethasone-treated MG-63 cells increased tartrate-resistant acid phosphatase (TRAP) activity by 54% (P < 0.005) compared with medium harvested from control-treated cells (in the presence of OPG-L and macrophage colony-stimulating factor). Moreover, dexamethasone (10−8m) promoted osteoclast formation in vitro, as assessed by a 2.5-fold increase of TRAP activity in cell lysates (P < 0.001) and the appearance of TRAP-positive multinucleated cells. Our data are thus consistent with the hypothesis that glucocorticoids promote osteoclastogenesis by inhibiting OPG and concurrently stimulating OPG-L production by osteoblastic lineage cells, thereby enhancing bone resorption.

Abstract Because regulation of the differentiation to osteoblasts and adipocytes from a common progenitor in bone marrow stroma is poorly understood, we assessed effects of bone morphogenetic protein-2 (BMP-2) on a conditionally immortalized human marrow stromal cell line, hMS(2–6), which is capable of differentiation to either lineage. BMP-2 did not affect hMS(2–6) cell proliferation but enhanced osteoblast differentiation as assessed by a 1.8-fold increase in expression of OSF2/CBFA1 (a gene involved in commitment to the osteoblast pathway), by increased mRNA expression and protein secretion for alkaline phosphatase (ALP), type I procollagen and osteocalcin (OC) (except for OC protein), and by increased mineralized nodule formation. Transient transfection with Osf2/Cbfa1 antisense oligonucleotide substantially reduced BMP-2–stimulated expression of ALP mRNA and protein. The effects of BMP-2 on adipocyte differentiation varied: expression of peroxisome proliferator-activated receptor γ2 (a gene involved in commitment to the adipocyte pathway) was unchanged, mRNA expression of the early differentiation marker, lipoprotein lipase, was increased, and mRNA and protein levels of the late differentiation marker, leptin, and the formation of cytoplasmic lipid droplets were decreased. Thus, by enhancing osteoblast commitment and by inhibiting late adipocyte maturation, BMP-2 acts to shunt uncommitted marrow stromal precursor cells from the adipocyte to the osteoblast differentiation pathway.

Abstract Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312–324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34°C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5°C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5°C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1α,25-dihydroxyvitamin D3 (1,25(OH2)D3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10−8 M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10−8 M 1,25(OH2)D3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH2)D3 were similar to changes in mRNA levels. When cultured at 39.5°C with ascorbate and β-glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH2)D3, or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans.