Lorenz C. Hofbauer

The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption

Although multiple hormones and cytokines regulate various aspects of osteoclast formation, the final two effectors are osteoprotegerin ligand (OPG‐L)/osteoclast differentiation factor (ODF), a recently cloned member of the tumor necrosis factor superfamily, and macrophage colony–stimulating factor. OPG‐L/ODF is produced by osteoblast lineage cells and exerts its biological effects through binding to its receptor, osteoclast differentiation and activation receptor (ODAR)/receptor activator of NF‐κB (RANK), on osteoclast lineage cells, in either a soluble or a membrane‐bound form, the latter of which requires cell‐to‐cell contact. Binding results in rapid differentiation of osteoclast precursors in bone marrow to mature osteoclasts and, at higher concentrations, in increased functional activity and reduced apoptosis of mature osteoclasts. The biological activity of OPG‐L/ODF is neutralized by binding to osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF), a member of the TNF‐receptor superfamily that also is secreted by osteoblast lineage cells. The biological importance of this system is underscored by the induction in mice of severe osteoporosis by targeted ablation of OPG/OCIF and by the induction of osteopetrosis by targeted ablation of OPG‐L/ODF or overexpression of OPG/OCIF. Thus, osteoclast formation may be determined principally by the relative ratio of OPG‐L/ODF to OPG/OCIF in the bone marrow microenvironment, and alterations in this ratio may be a major cause of bone loss in many metabolic disorders, including estrogen deficiency and glucocorticoid excess. That changes in but two downstream cytokines mediate the effects of large numbers of upstream hormones and cytokines suggests a regulatory mechanism for osteoclastogenesis of great efficiency and elegance.

New HorizonsOsteoporosis: now and the future

Osteoporosis is a common disease characterised by a systemic impairment of bone mass and microarchitecture that results in fragility fractures. With an ageing population, the medical and socioeconomic effect of osteoporosis, particularly postmenopausal osteoporosis, will increase further. A detailed knowledge of bone biology with molecular insights into the communication between bone-forming osteoblasts and bone-resorbing osteoclasts and the orchestrating signalling network has led to the identification of novel therapeutic targets. Novel treatment strategies have been developed that aim to inhibit excessive bone resorption and increase bone formation. The most promising novel treatments include: denosumab, a monoclonal antibody for receptor activator of NF-κB ligand, a key osteoclast cytokine; odanacatib, a specific inhibitor of the osteoclast protease cathepsin K; and antibodies against the proteins sclerostin and dickkopf-1, two endogenous inhibitors of bone formation. This overview discusses these novel therapies and explains their underlying physiology.

Stimulation of Osteoprotegerin Ligand and Inhibition of Osteoprotegerin Production by Glucocorticoids in Human Osteoblastic Lineage Cells: Potential Paracrine Mechanisms of Glucocorticoid-Induced Osteoporosis*

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 (10 210 M‐10 27 M) inhibited constitutive OPG protein concentrations in the conditioned medium of hFOB cells from 2.59 6 0.02 ng/ml (control) to 0.30 6 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 28 M) 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. (Endocrinology 140: 4382‐ 4389, 1999)

Interleukin-1β and tumor necrosis factor-α, but not interleukin-6, stimulate osteoprotegerin ligand gene expression in human osteoblastic cells

Abstract Recent studies have identified osteoprotegerin ligand (OPG-L) as the essential factor required for osteoclastogenesis, and that the effects are prevented by its soluble receptor, osteoprotegerin (OPG). However, there are limited data at present on the regulation of OPG-L expression in human osteoblastic cells by other cytokines. Because interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 all increase osteoclastogenesis, we assessed whether OPG-L mRNA steady-state levels were regulated by these cytokines in human osteoblastic cells. By northern analysis, IL-1β (5 nmol/L) and TNF-α (9 nmol/L) increased OPG-L mRNA steady-state levels by up to two- to three-fold in normal marrow stromal cells (MS), an immortalized marrow stromal cell line (hMS), and the osteosarcoma cell line, MG-63, whereas IL-6 (2 nmol/L, with or without its soluble receptor) had no effect on OPG-L mRNA levels in any of these cells. IL-1β and TNF-α increased OPG-L mRNA steady-state levels in the normal MS cells and the hMS cell line in a time- and dose-dependent fashion by up to 4.1-fold and up to 2.6-fold, respectively. Our data are thus consistent with the hypothesis that the proinflammatory and bone-resorbing cytokines, IL-1β and TNF-α, but not IL-6, may stimulate osteoclastogenesis by inducing the expression of OPG-L.

The Expression of Osteoprotegerin and RANK Ligand and the Support of Osteoclast Formation by Stromal- Osteoblast Lineage Cells Is Developmentally Regulated*

The one or more molecular mechanisms that determine the obligatory sequence of resorption followed by formation during bone remodeling is unclear. RANK ligand (RANK-L) is an essential requirement for osteoclastogenesis, and its activity is neutralized by binding to the soluble decoy receptor, osteoprotegerin (OPG). Because both molecules are produced by osteoblast lineage cells, we studied their developmental regulation in a conditionally immortalized human marrow stromal (hMS[2-15]) cell line. These cells can simulate the complete developmental sequence from undifferentiated precursor(s) to cells with the complete osteoblast phenotype that are capable of forming mineralized nodules. During osteoblast differentiation, RANK-L messenger RNA levels decreased by 5-fold, whereas OPG messenger RNA levels increased by 7-fold, resulting in a 35-fold change in the RANK-L/OPG ratio. OPG protein also increased by 6-fold. Mouse bone marrow cells generated osteoclast-like cells in coculture with undifferentiated hMS(2-15) cells, but did not when cocultured with hMS(2-15) cells in varying stages of differentiation, unless an excess of RANK-L was added. Thus, undifferentiated marrow stromal cells with a high RANK-L/OPG ratio can initiate and support osteoclastogenesis, but after differentiation to the mature osteoblast phenotype, they cannot. We speculate that the developmental regulation of OPG and RANK-L production by stromal/osteoblast cells contributes to the coordinated sequence of osteoclast and osteoblast differentiation during the bone remodeling cycle.

Phytoestrogen genistein stimulates the production of osteoprotegerin by human trabecular osteoblasts.

The anti‐resorptive effects of estrogen on bone metabolism are thought to be mediated through modulation of paracrine factors produced by osteoblastic lineage cells that act on osteoclastic lineage cells. Receptor activator of nuclear factor‐κB ligand (RANKL) is the essential factor for osteoclast formation and activation and enhances bone resorption. By contrast, osteoprotegerin (OPG), which is produced by osteoblastic lineage cells acts as a decoy receptor that neutralizes RANKL and prevents bone loss. Recently, 17β‐estradiol was found to stimulate OPG mRNA levels and protein secretion in a human osteoblastic cell line through activation of the estrogen receptor (ER)‐α. In this study, we assessed the effects of the phytoestrogen genistein on OPG mRNA steady state levels (by semiquantitative RT‐PCR and Northern analysis) and protein production (by ELISA) in primary human trabecular osteoblasts (hOB) obtained from healthy donors. Genistein increased OPG mRNA levels and protein secretion by hOB cells by up to two‐ to six‐fold in a dose‐ (Pu2009<u20090.0001) and time‐dependent (Pu2009<u20090.0001) fashion with a maximum effect at 10−7 M. Co‐treatment with the pure ER antagonist ICI 182,780 completely abrogated the stimulatory effects of genistein on OPG protein secretion, indicating that these effects were specific and directly mediated through the ER. Pre‐treatment with genistein partially prevented the inhibitory effects of the glucocorticoid dexamethasone on OPG mRNA and protein production. The stimulation of OPG mRNA levels by genistein was not affected by the protein synthesis inhibitor, cycloheximide and was shown to be due to enhancement of OPG gene transcription. In conclusion, our data suggest that the phytoestrogen genistein is capable of upregulating the production of OPG by human osteoblasts. Thus, dietary sources of phytoestrogens may help to prevent bone resorption and bone loss by enhanced osteoblastic production of OPG. J. Cell. Biochem. 84: 725–735, 2002.

Bone morphogenetic protein-6 production in human osteoblastic cell lines. Selective regulation by estrogen.

Bone morphogenetic proteins (BMPs) induce differentiation of osteoblast and chondroblast lineage cells from uncommitted mesenchymal precursors. Because estrogen has potent osteochondrogenic actions, we investigated its effect on BMP production in two estrogen-responsive, human immortalized cell lines (hFOB/ER3 and hFOB/ER9) that display the mature osteoblast phenotype. These cell lines were produced by stable transfection of the estrogen receptor (ER) gene into immortalized fetal osteoblasts at low ( approximately 800 ER/ nucleus) and at high ( approximately 3, 900 ER/nucleus) levels, respectively. As assessed by reverse transcriptase PCR, treatment with 17beta-estradiol (10(-)10 - 10(-)7 M) increased steady-state levels of BMP-6 mRNA dose dependently by twofold in the hFOB/ER3 cells and by over threefold in the hFOB/ER9 cells. Messenger RNA levels for transforming growth factors-beta1 and -beta2 and BMPs-1 through -5 and -7 levels were unchanged. The results were confirmed by sequence determination of the PCR product and by Northern blot analysis for total RNA. 17beta-estradiol increased BMP-6 protein production sixfold by Western analysis. Cotreatment with antiestrogens (ICI 182,780 or 4-hydroxytamoxifen) antagonized the effects of 17beta-estradiol. These data suggest that some of the skeletal effects of estrogen on bone and cartilage may be mediated by increased production of BMP-6 by osteoblasts.

Gorham-Stout Disease—Stabilization During Bisphosphonate Treatment†

A 45‐year‐old woman presented with recent onset of left‐sided chest pain. On clinical examination, these symptoms seemed to be strictly localized to a region that was marked by a long‐standing cutaneous erythematous lesion. Laboratory results showed no gross abnormalities. Radiological imaging including conventional X‐ray, MRI scans, and 3D CT reconstruction of the rib cage revealed circumscript destruction of the left lateral ribs 9–11. Histological analysis of a rib biopsy showed angiomatous hypervascularization and intracortical fibrosis. In keeping with these findings, the patients condition was diagnosed as Gorham‐Stout disease, a rare condition with localized, often unilateral, bone destruction. Monotherapy with bisphosphonates (pamidronate 30 mg IV every 3 months) was initiated, leading to rapid disappearance of local pain. Follow‐up over 24 months documented a stable clinical and radiological picture without evidence of progressive bone destruction.

The Anti‐Androgen Hydroxyflutamide and Androgens Inhibit Interleukin‐6 Production by an Androgen‐Responsive Human Osteoblastic Cell Line

While androgens clearly have significant skeletal effects, the paracrine mediators of androgen action on bone are at present unclear. Interleukin‐6 (IL‐6) is a candidate cytokine that is produced by osteoblastic lineage cells and promotes osteoclastogenesis and bone resorption. Here, we assessed constitutive as well as IL‐1β– and tumor necrosis factor‐α (TNF‐α)–stimulated IL‐6 mRNA expression by Northern analysis and protein secretion by immunoassay in a human androgen‐responsive osteoblastic cell line (hFOB/AR‐6) which contains ∼4000 androgen receptors (ARs)/nucleus. Treatment with 5α‐dihydrotestosterone (DHT) dose‐dependently inhibited constitutive and TNF‐α/IL‐1β–stimulated IL‐6 mRNA steady‐state levels in hFOB/AR‐6 cells by 70–80% at 10−7 M. In addition, testosterone also suppressed TNF‐α/IL‐1β–stimulated IL‐6 mRNA levels by 57%, while the adrenal androgen dehydroepiandrosterone had no effect. Of note, the specific AR antagonist, hydroxyflutamide, also inhibited IL‐6 mRNA levels by 70%. Consistent with the Northern analyses, treatment with 5α‐DHT, testosterone, and hydroxyflutamide also inhibited IL‐6 protein production by 79%, 62%, and 71%, respectively (p < 0.001), while these agents had no effect on IL‐6 soluble receptor levels. Finally, we demonstrated that hydroxyflutamide treatment of hFOB/AR‐6 cells markedly inhibited the activation and binding of NF‐κB (a known stimulator of IL‐6 gene transcription) to its response element, thus providing a potential mechanism for its effect on IL‐6 production by osteoblasts. These data are consistent with the hypothesis that suppression of osteoblast IL‐6 production by androgens may mediate, at least in part, the antiresorptive effects of androgens on bone. Moreover, our findings also indicate that hydroxyflutamide, which is a known AR antagonist in most tissues, may function as a selective AR modulator for effects on IL‐6 production by osteoblasts.

Effects of gonadal and adrenal androgens in a novel androgen-responsive human osteoblastic cell line.

While androgens have important skeletal effects, the mechanism(s) of androgen action on bone remain unclear. Current osteoblast models to study androgen effects have several limitations, including the presence of heterogeneous cell populations. In this study, we examined the effects of androgens on the proliferation and differentiation of a novel human fetal osteoblastic cell line (hFOB/AR‐6) that expresses a mature osteoblast phenotype and a physiological number (∼4,000/nucleus) of androgen receptors (AR). Treatment with 5α‐dihydrotestosterone (5α‐DHT) inhibited the proliferation of hFOB/AR‐6 cells in a dose‐dependent fashion, while it had no effect on the proliferation of hFOB cells, which express low levels of AR (<200/nucleus). In hFOB/AR‐6 cells, co‐treatment with the specific AR antagonist, hydroxyflutamide abolished 5α‐DHT‐induced growth inhibition. Steady‐state levels of transforming growth factor‐β1 (TGF‐β1) and TGF‐β‐induced early gene (TIEG) mRNA decreased after treatment of hFOB/AR‐6 cells with 5α‐DHT, suggesting a role for the TGF‐β1‐TIEG pathway in mediating 5α‐DHT‐induced growth inhibition of hFOB/AR‐6 cells. In support of this, co‐treatment of hFOB/AR‐6 cells with TGF‐β1 (40 pg/ml) reversed the 5α‐DHT‐induced growth inhibition, whereas TGF‐β1 alone at this dose had no effect on hFOB/AR‐6 cell proliferation. Furthermore, treatment of hFOB/AR‐6 cells with 5α‐DHT and testosterone (10−8 M) inhibited basal and 1,25‐(OH)2D3‐induced alkaline phosphatase (ALP) activity and type I collagen synthesis without affecting osteocalcin production. Thus, in this fetal osteoblast cell line expressing a physiological number of AR, androgens decrease proliferation and the expression of markers associated with osteoblast differentiation. These studies suggest that the potential anabolic effect of androgens on bone may not be mediated at the level of the mature osteoblast. J. Cell. Biochem. 71:96–108, 1998.