Rapid CommunicationsInterleukin-1β and tumor necrosis factor-α, but not interleukin-6, stimulate osteoprotegerin ligand gene expression in human osteoblastic cells
Introduction
Enhanced osteoclastogenesis is a hallmark of various forms of metabolic bone diseases, including postmenopausal and glucocorticoid-induced osteoporosis, and leads to increased bone resorption and bone loss. In the bone microenvironment, osteoclast differentiation is regulated by the coordinated synthesis and action of cytokines produced by bone marrow stromal cells and osteoblasts.22, 25 Recently, osteoprotegerin ligand (OPG-L) has been identified as a novel member of the tumor necrosis factor (TNF) ligand family.1, 20, 36, 37 Independent in vitro studies have demonstrated that OPG-L, in the presence of macrophage-stimulating factor (M-CSF), is both necessary and sufficient for complete osteoclastogenesis from multipotential osteoclast precursor cells to fully committed, mature osteoclasts.20, 26, 37 Moreover, OPG-L administration to normal mice resulted in enhanced osteoclastogenesis, severe osteoporosis, and malignant hypercalcemia in vivo.20 The soluble receptor, osteoprotegerin (OPG), a member of the TNF receptor (TNF-R) superfamily,19, 28, 30, 38 neutralizes the effects of OPG-L.8, 20, 26, 37 Overexpression of OPG in transgenic mice resulted in osteopetrosis (generalized increased bone mass), and the administration of OPG to normal animals prevented ovariectomy-induced bone loss.28 In contrast, targeted ablation of the OPG gene in knockout mice resulted in unopposed actions of OPG-L, and to early-onset, severe osteoporosis.5, 23
Because OPG-L, in the presence of M-CSF, is the only cytokine required for osteoclastogenesis in vitro, and genetic manipulation of this system resulted in both extremes of skeletal phenotype (osteopetrosis, osteoporosis) in vivo, it has been suggested that other calcitropic hormones and cytokines may regulate osteoclastogenesis and bone resorption by modulating the expression of OPG-L and OPG by osteoblastic cells.11 Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6 are key bone-resorbing cytokines that have also been implicated in the pathogenesis of postmenopausal bone loss.15, 22, 25 Although these cytokines clearly increase osteoclastogenesis,15, 22, 25 it is unclear whether these effects are mediated by increased OPG-L production by cells in the bone microenvironment or are independent of them. To test this, we assessed the effects of these cytokines on OPG-L mRNA steady-state levels by northern analysis in human osteoblastic and bone marrow stromal cells.
Section snippets
Materials
Culture flasks and dishes were obtained from Corning (Corning, NY). Cell culture medium and supplements were purchased from Sigma (St. Louis, MO). The random primer labeling kit (Decaprime II) was from Ambion (Austin, TX) and [α-32P]-dCTP was from DuPont-NEN (Boston, MA). The human β-actin cDNA insert and ExpressHyb solution were obtained from Clontech (Palo Alto, CA). Recombinant human TNF-α, IL-1β, IL-6, and IL-6 soluble receptor (IL-6sR) were from R&D Systems (Minneapolis, MN).
Cell cultures
The following
Results
IL-1β (5 nmol/L) and TNF-α (9 nmol/L) increased OPG-L mRNA steady-state levels (a single OPG-L mRNA species of 2.4 kb) by 2.2-fold and 1.9-fold, respectively, in the differentiated osteosarcoma cell line, MG-63 (Figure 1, lanes 2 and 3). In contrast, IL-6 (nmol/L) had no effect on OPG-L mRNA steady-state levels either without or with IL-6sR, which may be required for appropriate IL-6 action (Figure 1, lanes 4 and 5).24 Rehybridization of the membrane with the radiolabeled OPG cDNA probe
Discussion
The balance between bone resorption and bone formation is regulated by an integrated network of cytokines that includes macrophage colony-stimulating factor (M-CSF), IL-1, IL-4, IL-6, IL-11, prostaglandin estradiol (PGE2), TNF-α, and transforming growth factor-β (TGF-β), which are synthesized by bone marrow stromal cells and osteoblasts and which modulate osteoclast differentiation and activity.15, 22, 25 Although there is consensus about the role of this cytokine network, the importance of one
Acknowledgements
The authors acknowledge the technical assistance of M. J. Schroeder, B. Ngo, and R. A. Soderberg. This work was supported by Grant AG-04875 from the National Institutes of Health. Dr. Hofbauer is a recipient of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (Ho 1875/1-1).
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