Atorvastatin stimulates the production of osteoprotegerin by human osteoblasts

Recently, HMG-CoA reductase inhibitors (statins), potent inhibitors of cholesterol biosynthesis, have been linked to protective effects on bone metabolism. Because of their widespread use, prevention of bone loss and fractures would be a desirable side effect. However, the mechanisms how statins may affect bone metabolism are poorly defined. Here, we evaluated the effect of atorvastatin on osteoblastic production of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG), cytokines that are essential for osteoclast cell biology. While RANKL enhances osteoclast formation and activation, thereby, promoting bone loss, OPG acts as a soluble decoy receptor and antagonizes the effects of RANKL. In primary human osteoblasts (hOB), atorvastatin increased OPG mRNA levels and protein secretion by hOB by up to three fold in a dose-dependent manner with a maximum effect at 10(-6) M (P < 0.001). Time course experiments indicated a time-dependent stimulatory effect of atorvastatin on OPG mRNA levels after 24 h and on OPG protein secretion after 48-72 h (P < 0.001). Treatment of hOB with substrates of cholesterol biosynthesis that are downstream of the HMG-CoA reductase reaction (mevalonate, geranylgeranyl pyrophosphate) reversed atorvastatin-induced enhancement of OPG production. Of note, atorvastatin abrogated the inhibitory effect of glucocorticoids on OPG production. Treatment of hOB with atorvastatin enhanced the expression of osteoblastic differentiation markers, alkaline phosphatase and osteocalcin. In summary, our data suggest that atorvastatin enhances osteoblastic differentiation and production of OPG. This may contribute to the bone-sparing effects of statins.     

Effects of arachidonic acid, docosahexaenoic acid, prostaglandin E(2) and parathyroid hormone on osteoprotegerin and RANKL secretion by MC3T3-E1 osteoblast-like cells

Bone is continuously remodeled through resorption by osteoclasts and the subsequent synthesis of the bone matrix by osteoblasts. Cell-to-cell contact between osteoblasts and osteoclast precursors is required for osteoclast formation. RANKL (receptor activator of nuclear factor-kappaB ligand) expressed on osteoblastic cell membranes stimulates osteoclastogenesis, while osteoprotegerin (OPG) secreted by osteoblasts inhibits osteoclastogenesis. Although polyunsaturated fatty acids (PUFAs) have been implicated in bone homeostasis, the effects thereof on OPG and RANKL secretion have not been investigated. MC3T3-E1 osteoblasts were exposed to the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA); furthermore, the bone-active hormone parathyroid hormone (PTH) and the effects thereof were tested on OPG and RANKL secretion. Prostaglandin E(2) (PGE(2)), a product of AA metabolism that was previously implicated in bone homeostasis, was included in the study. AA (5.0-20 microg/ml) inhibited OPG secretion by 25-30%, which was attenuated by pretreatment with the cyclooxygenase blocker indomethacin, suggesting that the inhibitory effect of AA on OPG could possibly be PGE(2)-mediated. MC3T3-E1 cells secreted very low basal levels of RANKL, but AA stimulated RANKL secretion, thereby decreasing the OPG/RANKL ratio. DHA suppressed OPG secretion to a smaller extent than AA. This could, however, be due to endogenous PGE(2) production. No RANKL could be detected after exposing the MC3T3-E1 cells to DHA. PTH did not affect OPG secretion, but stimulated RANKL secretion. This study demonstrates that AA and PTH reduce the OPG/RANKL ratio and may increase osteoclastogenesis. DHA, however, had no significant effect on OPG or RANKL in this model.     

Metformin stimulates osteoprotegerin and reduces RANKL expression in osteoblasts and ovariectomized rats

Anti-diabetic drug metformin has been shown to enhance osteoblasts differentiation and inhibit osteoclast differentiation in vitro and prevent bone loss in ovariectomized (OVX) rats. But the mechanisms through which metformin regulates osteoclastogensis are not known. Osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) are cytokines predominantly secreted by osteoblasts and play critical roles in the differentiation and function of osteoclasts. In this study, we demonstrated that metformin dose-dependently stimulated OPG and reduced RANKL mRNA and protein expression in mouse calvarial osteoblasts and osteoblastic cell line MC3T3-E1. Inhibition of AMP-activated protein kinase (AMPK) and CaM kinase kinase (CaMKK), two targets of metformin, suppressed endogenous and metformin-induced OPG secretion in osteoblasts. Moreover, supernatant of osteoblasts treated with metformin reduced formation of tartrate resistant acid phosphatase (TRAP)-positive multi-nucleated cells in Raw264.7 cells. Most importantly, metformin significantly increased total body bone mineral density, prevented bone loss and decreased TRAP-positive cells in OVX rats proximal tibiae, accompanied with an increase of OPG and decrease of RANKL expression. These in vivo and in vitro studies suggest that metformin reduces RANKL and stimulates OPG expression in osteoblasts, further inhibits osteoclast differentiation and prevents bone loss in OVX rats.     

CGRP inhibits osteoprotegerin production in human osteoblast-like cells via cAMP/PKA-dependent pathway

The osteoprotegerin (OPG)/receptor activator of nuclear factor-B ligand (RANKL)/receptor activator of nuclear factor-B (RANK) system was evaluated as a potential target of CGRP anabolic activity on bone. Primary cultures of human osteoblast-like cells (hOB) express calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1, and, because CGRP stimulates cAMP (one of the modulators of OPG production in osteoblasts), it was investigated whether it affects OPG secretion and expression in hOB. CGRP treatment of hOB (10^–11 M–10^–7 M) dose-dependently inhibited OPG secretion with an EC₅₀ of 1.08 x 10^–10 M, and also decreased its expression. This action was blocked by the antagonist CGRP_8–37. Forskolin, a stimulator of cAMP production, and dibutyryl cAMP also reduced the production of OPG. CGRP (10^–8 M) enhanced protein kinase A (PKA) activity in hOB, and hOB exposure to the PKA inhibitor, H89 (2 x 10^–6 M), abolished the inhibitory effect of CGRP on OPG secretion. Conditioned media from CGRP-treated hOB increased the number of multinucleated tartrate-resistant acid phosphatase-positive cells and the secretion of cathepsin K in human peripheral blood mononuclear cells compared with the conditioned media of untreated hOB. These results show that the cAMP/PKA pathway is involved in the CGRP inhibition of OPG mRNA and protein secretion in hOB and that this effect favors osteoclastogenesis. CGRP could thus modulate the balance between osteoblast and osteoclast activity, participating in the fine tuning of all of the bone remodeling phases necessary for the subsequent anabolic effect. receptor-activity-modifying proteins; protein kinase A; osteoclast; cathepsin K     

Effects of immunosuppressants on receptor activator of NF-kappaB ligand and osteoprotegerin production by human osteoblastic and coronary artery smooth muscle cells

Osteoporosis and vasculopathy are common after organ transplantation and have been largely attributed to the use of immunosuppressants. Osteoprotegerin (OPG) is produced by osteoblastic and arterial cells, and inhibits osteoclast functions by neutralizing receptor activator of NF-kappaB ligand (RANKL). Because OPG-deficient mice develop osteoporosis and arterial calcification, we assessed the effects of immunosuppressants on OPG and RANKL expression by human osteoblastic and coronary artery smooth muscle cells (CASMC). Cyclosporine A, rapamycin, and FK-506 decreased OPG mRNA and protein levels in undifferentiated marrow stromal cells (by 63, 44, and 68%, respectively, P < 0.001). All three immunosuppressants increased RANKL mRNA levels in these cells by 60 to 210%. In contrast to these effects on marrow stromal cells, rapamycin, which may be relatively bone-sparing, increased OPG mRNA and protein production (by 120%, P < 0.001) in mature osteoblastic cells. Cyclosporine A also decreased OPG mRNA and protein production (by 52%, P < 0.001) of CASMC. In conclusion, immunosuppressants decrease OPG mRNA and protein production and increase RANKL gene expression by marrow stromal cells, and cyclosporine suppresses OPG production in CASMC. These studies thus provide a potential mechanism for immunosuppressant-induced bone loss, and the propensity of cyclosporine A to cause vascular disease.     

Effects of different magnitudes of mechanical strain on Osteoblasts in vitro

In addition to systemic and local factors, mechanical strain plays a crucial role in bone remodeling during growth, development, and fracture healing, and especially in orthodontic tooth movement. Although many papers have been published on the effects of mechanical stress on osteoblasts or osteoblastic cells, little is known about the effects of different magnitudes of mechanical strain on such cells. In the present study, we investigated how different magnitudes of cyclic tensile strain affected osteoblasts. MC3T3-E1 osteoblastic cells were subjected to 0%, 6%, 12% or 18% elongation for 24h using a Flexercell Strain Unit, and then the mRNA and protein expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) were examined. The results showed that cyclic tensile strain induced a magnitude-dependent increase (0%, 6%, 12%, and 18%) in OPG synthesis and a concomitant decrease in RANKL mRNA expression and sRANKL release from the osteoblasts. Furthermore, the induction of OPG mRNA expression by stretching was inhibited by indomethacin or genistein, and the stretch-induced reduction of RANKL mRNA was inhibited by PD098059. These results indicate that different magnitudes of cyclic tensile strain influence the biological behavior of osteoblasts, which profoundly affects bone remodeling.     

The OPG/RANKL/RANK system in metabolic bone diseases

The OPG/RANKL/RANK cytokine system is essential for osteoclast biology. Various studies suggest that human metabolic bone diseases are related to alterations of this system. Here we summarize OPG/RANKL/RANK abnormalities in different forms of osteoporoses and hyperparathyroidism. Skeletal estrogen agonists (including 17beta-estradiol, raloxifene, and genistein) induce osteoblastic OPG production through estrogen receptor-alpha activation in vitro, while immune cells appear to over-express RANKL in estrogen deficiency in vivo. Of note, OPG administration can prevent bone loss associated with estrogen deficiency as observed in both animal models and a small clinical study. Glucocorticoids and immunosuppressants concurrently up-regulate RANKL and suppress OPG in osteoblastic cells in vitro, and glucocorticoids are among the most powerful drugs to suppress OPG serum levels in vivo. As for mechanisms of immobilization-induced bone loss, it appears that mechanical strain inhibits RANKL production through the ERK 1/2 MAP kinase pathway and up-regulates OPG production in vitro. Hence, lack of mechanical strainduring immobilization may favor an enhanced RANKL-to-OPG ratio leading to increased bone loss. As for hyperparathyroidism, chronic PTH exposure concurrently enhances RANKL production and suppresses OPG secretion through activation of osteoblastic protein kinase A in vitro which would favour increased osteoclastic activity. In sum, the capacity for OPG to antagonize the increases in bone loss seen in many rodent models of metabolic bone disease implicates RANKL/OPG imbalances as the likely etiology and supports the potential role for a RANKL antagonist as a therapeutic intervention in these settings.     

Zoledronate promotes osteoblast differentiation in high-glucose conditions via the p38MAPK pathway

Zoledronate (ZOL) were found to inhibit bone resorption in an animal model of diabetes, high glucose concentrations have been shown to decreased the osteogenesis-related gene expression. But the molecular mechanism by which high glucose levels affect osteoblasts and the effects of ZOL on osteoblast differentiation in a high-glucose environment remain unclear. Therefore, we aimed to investigate the effect of ZOL on osteoblast differentiation in a high-glucose environment and determine the responsible mechanism. Cell proliferation was detected by MTT assay, and cell differentiation was evaluated by immunofluorescence staining for alkaline phosphatase expression, alizarin red staining, cytoskeletal arrangement, and actin fiber formation. Real-time PCR and western blot analyses were performed to detect the mRNA and protein expression of p38MAPK, phosphorylated (p)-p38MAPK, CREB, p-CREB, collagen (COL) I, osteoprotegerin (OPG), and RANKL. The results showed that cell proliferation activity did not differ among the groups. But high glucose inhibited osteoblast differentiation; actin fiber formation; and p38MAPK, p-p38MAPK, CREB, p-CREB, COL I, and OPG expression, while promoting RANKL expression. However, we found that treatment with ZOL reversed these effects of high glucose. And further addition of a p38MAPK inhibitor led to inhibition of osteoblast differentiation and actin fiber formation, and lower p38MAPK, p-p38MAPK, CREB, p-CREB, COL I, and OPG expression than in the high glucose +ZOL group with higher RANKL expression than in the high glucose +ZOL group. Collectively, this study demonstrates that high glucose inhibits the differentiation of osteoblasts, and ZOL could partly overcome these effects by regulating p38MAPK pathway activity.     

Osteogenic growth peptide effects on primary human osteoblast cultures: potential relevance for the treatment of glucocorticoid-induced osteoporosis

The osteogenic growth peptide (OGP) is a naturally occurring tetradecapeptide that has attracted considerable clinical interest as a bone anabolic agent and hematopoietic stimulator. In vivo studies on animals have demonstrated that the synthetic peptide OGP (10-14), reproducing the OGP C-terminal active portion [H-Tyr-Gly-Phe-Gly-Gly-OH] increases bone formation, trabecular bone density and fracture healing. In vitro studies performed on cellular systems based on osteoblastic-like cell lines or mouse stromal cells, have demonstrated that OGP (10-14) increases osteoblast proliferation, alkaline phosphatase (ALKP) activity and matrix synthesis and mineralization. In view of a potential application of OGP (10-14) in clinical therapy, we have tested different concentrations of OGP (10-14) on primary human osteoblast (hOB) cultures. We have observed significant increases of hOB proliferation (+35%), ALKP activity (+60%), osteocalcin secretion (+50%), and mineralized nodules formation (+49%). Our experimental model based on mature hOBs was used to investigate if OGP (10-14) could prevent the effects on bone loss induced by sustained glucocorticoid (GC) treatments. A strong decrease in bone formation has been attributed to the effects of GCs on osteoblastogenesis and osteocyte apoptosis, while an increase in bone resorption was due to a transient osteoblastic stimulation, mediated by the OPG/RANKL/RANK system, of osteoclasts recruitment and activation. Moreover, GCs act on hOBs decreasing the release of osteoprotegerin (OPG) a regulator of the RANKL/RANK interaction. Here, we provide evidences that OGP (10-14) inhibits hOB apoptosis induced by an excess of dexamethasone (-48% of apoptotic cells). Furthermore, we show that OGP (10-14) can increase OPG secretion (+20%) and can restore the altered expression of OPG induced by GCs to physiological levels. Our results support the employment of OGP (10-14) in clinical trials addressed to the treatment of different bone remodeling alterations including the GC-induced osteoporosis.     

IL-13 regulates vascular cell adhesion molecule-1 expression in human osteoblasts

Activated T cells (Act T) produce multiple cytokines that affect osteoblast function as well as osteoclastogenesis. One of these cytokines, IL-13, is a multifunctional cytokine elaborated by Act T that regulates vascular cellular adhesion molecule (VCAM)-1 expression in endothelial cells. VCAM-1 has also been implicated in osteoclast formation by myeloma cells. We therefore studied whether IL-13 regulates VCAM-1 in human osteoblastic cells since these cells express RANKL, the major osteoclastogenic factor and osteoclast precursors are found adjacent to osteoblasts. Human T cells were activated in the absence or presence of Cyclosporin A (CsA), an inhibitor of the production of most activated T cell cytokines. Conditioned media were assayed for IL-13 by ELISA. Act T produced IL-13 and, unlike other T cell cytokines, this was elevated 3-fold by CsA. Exposure of human osteoblasts (hOB) to doses of recombinant human IL-13 (rhIL-13, 0-10 ng/ml) resulted in an increase of VCAM-1 mRNA (up to 5-fold) within 4 h with a maximum stimulation at 1 ng/ml. CsA had no effect on basal hOB VCAM-1 mRNA expression. Examination of VCAM-1 on the cell surface of hOB, by immunocytochemistry, revealed increasing levels of surface expression of the protein within 16 h after stimulation with doses of rhIL-13 (0.1-10 ng/ml) which were reflective of the mRNAs. IL-6 production was also stimulated in a dose dependent manner with a maximum of 2.5-fold with 1 ng/ml rhIL-13 within 16 h. Since both VCAM-1 and IL-6 showed similar responses to IL-13, IL-6 was examined for its ability to induce VCAM-1. Immunocytochemistry demonstrated no effect of IL-6 on VCAM-1 expression. These data demonstrate that during pathological processes associated with T cell activation, such as rheumatoid arthritis or possibly post-menopausal osteoporosis, T cells may play a pivotal role in osteoclast precursor adhesion to osteoblasts as a first step prior to RANKL signaling.     

Suppression of osteoprotegerin expression by prostaglandin E2 is crucially involved in lipopolysaccharide-induced osteoclast formation

LPS is a potent stimulator of bone resorption in inflammatory diseases. The mechanism by which LPS induces osteoclastogenesis was studied in cocultures of mouse osteoblasts and bone marrow cells. LPS stimulated osteoclast formation and PGE(2) production in cocultures of mouse osteoblasts and bone marrow cells, and the stimulation was completely inhibited by NS398, a cyclooxygenase-2 inhibitor. Osteoblasts, but not bone marrow cells, produced PGE(2) in response to LPS. LPS-induced osteoclast formation was also inhibited by osteoprotegerin (OPG), a decoy receptor of receptor activator of NF-kappaB ligand (RANKL), but not by anti-mouse TNFR1 Ab or IL-1 receptor antagonist. LPS induced both stimulation of RANKL mRNA expression and inhibition of OPG mRNA expression in osteoblasts. NS398 blocked LPS-induced down-regulation of OPG mRNA expression, but not LPS-induced up-regulation of RANKL mRNA expression, suggesting that down-regulation of OPG expression by PGE(2) is involved in LPS-induced osteoclast formation in the cocultures. NS398 failed to inhibit LPS-induced osteoclastogenesis in cocultures containing OPG knockout mouse-derived osteoblasts. IL-1 also stimulated PGE(2) production in osteoblasts and osteoclast formation in the cocultures, and the stimulation was inhibited by NS398. As seen with LPS, NS398 failed to inhibit IL-1-induced osteoclast formation in cocultures with OPG-deficient osteoblasts. These results suggest that IL-1 as well as LPS stimulates osteoclastogenesis through two parallel events: direct enhancement of RANKL expression and suppression of OPG expression, which is mediated by PGE(2) production.     

Mechanical stimulation effects on functional end effectors in osteoblastic MG-63 cells

Receptor activator of Nf-kappaB ligand (RANKL) and osteoprotegerin (OPG) have been implicated in bone metabolism. Specifically, the balance of these factors in conjunction with receptor activator of Nf-kappaB (RANK) is believed to be key in determining the rate of osteoclastogenesis and the net outcome of bone formation/resorption. While it is well accepted that mechanical loading in vivo affects bone formation/resorption and that alterations in the responsiveness of bone cells to mechanical loading have been implicated in metabolic bone diseases, the effect of in vitro mechanical loading on osteoblastic production of OPG and RANKL has not been extensively studied. Thus, in the current study, we developed an in vitro model to load human osteoblasts and studied levels of OPG, RANKL, PGE(2) and macrophage colony stimulating factor (M-CSF). We hypothesized that stimulating osteoblastic cells would increase the release of soluble OPG relative to RANKL favoring a bone-forming (and resorption-inhibiting) event. To accomplish this, we developed a small-scale loading machine that imparts via bending, well-defined substrate deformation to bone cells cultured on artificial substrates. Following 2h of loading and a 1h incubation period, media was collected and levels of soluble OPG, RANKL, PGE(2) and M-CSF were quantified using ELISA and western blotting. We found that mechanical loading significantly increased soluble OPG levels relative to RANKL at this 3h time point. Levels of soluble and cellular RANKL detected were not significantly affected by mechanical stimulation. The relative shift in abundance of OPG over RANKL associated with applied mechanical stimulation suggests the soluble OPG:RANKL ratio may be important in load-induced coupling mechanisms of bone cells.     

Detection of osteoprotegerin (OPG) and its ligand (RANKL) mRNA and protein in femur and tibia of the rat

Osteoprotegerin (OPG) and the receptor activator of nuclear factor (NF)-kB ligand (RANKL) are key regulators of osteoclastogenesis. The present study had the main aim of showing the localization of OPG and RANKL mRNA and protein in serial sections of the rat femurs and tibiae by immunohistochemistry (IHC) and in situ hybridization (ISH). The main results were: (1) OPG and RANKL mRNA and protein were co-localized in the same cell types, (2) maturative/hypertrophic chondrocytes, osteoblasts, lining cells, periosteal cells and early osteocytes were stained by both IHC and ISH, (3) OPG and RANKL proteins were mainly located in Golgi areas, and the ISH reaction was especially visible in active osteoblasts, (4) immunolabeling was often concentrated into cytoplasmic vacuoles of otherwise negative proliferative chondrocytes; IHC and ISH labeling increased from proliferative to maturative/hypertrophic chondrocytes, (5) the newly laid down bone matrix, cartilage-bone interfaces, cement lines, and trabecular borders showed light OPG and RANKL immunolabeling, (6) about 70% of secondary metaphyseal bone osteocytes showed OPG and RANKL protein expression; most of them were ISH-negative, (7) osteoclasts were mostly unstained by IHC and variably labeled by ISH. The co-expression of OPG and RANKL in the same bone cell types confirms their strictly coupled action in the regulation of bone metabolism.     

The neuropeptide VIP regulates the expression of osteoclastogenic factors in osteoblasts

Osteoclast formation is controlled by stromal cells/osteoblasts expressing macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), crucial for osteoclast progenitor cell proliferation, survival and differentiation, and osteoprotegerin (OPG) that inhibits the interaction between RANKL and its receptor RANK. Recent data have strongly indicated that the nervous system plays an important role in bone biology. In the present study, the effects of the neuropeptide vasoactive intestinal peptide (VIP), present in peptidergic skeletal nerve fibers, on the expression of RANKL, OPG, and M-CSF in osteoblasts and stromal cells have been investigated. VIP and pituitary adenylate cyclase-activating polypeptide 38 (PACAP-38), but not secretin, stimulated rankl mRNA expression in mouse calvarial osteoblasts. In contrast, VIP inhibited the mRNA expressions of opg and m-csf, effects shared by PACAP-38, but not by secretin. VIP did not affect rankl, opg, or m-csf mRNA expression in mouse bone marrow stromal cells (BMSCs). The effects by VIP on the mRNA expression of rankl, opg, and m-csf were all potentiated by the cyclic AMP phosphodiesterase inhibitor rolipram. In addition, VIP robustly enhanced the phosphorylation of ERK and the stimulatory effect by VIP on rankl mRNA was inhibited by the MEK1/2 inhibitor PD98059. These observations demonstrate that activation of VPAC(2) receptors in osteoblasts enhances the RANKL/OPG ratio by mechanisms mediated by cyclic AMP and ERK pathways suggesting an important role for VIP in bone remodeling.     

Direct effects of osteoprotegerin on human bone cell metabolism

Purpose

Osteoprotegerin (OPG) affects bone metabolism by intercepting the RANK-RANKL interaction which prevents osteoclastic differentiation and consequently reduces bone resorption. Different bone phenotypes of mice overexpressing OPG and of mice with knockdown of receptor activator of NF-κB (RANK) or RANK-ligand (RANKL) suggest that the mechanism of action of the OPG-RANKL-RANK system in regulating bone remodeling is not completely understood. Furthermore, OPG increases bone mass and density independently from reduced osteoclastogenesis which is consistent with the possibility that OPG may directly affect bone metabolism beyond its known role as decoy receptor for RANKL.

Methods

We treated primary human osteoblastic cells with OPG and inhibitory anti-RANKL antibodies and measured cellular ALP activity, in vitro mineralization, vitronectin receptor protein expression and ERK phosphorylation. We also analyzed the mRNA co-expression of ALP and OPG ex vivo in bone biopsies from acute and old stable vertebral fractures.

Results

OPG directly increased ALP activity and in vitro mineralization of HOC, enhanced expression of the vitronectin receptor thereby increasing adherence of HOC to vitronectin and stimulated ERK phosphorylation. All OPG-mediated effects could be prevented by RANKL antibodies or RANKL-siRNA transfection and MAPK inhibitor PD98059 reduced the stimulatory effect of OPG on integrin αv expression. In acutely fractured vertebrae OPG and ALP mRNA expression was significantly increased compared to stable vertebral fractures. In conclusion, OPG exerts direct osteoanabolic effects on HOC metabolism via RANKL in addition to its well described role as decoy receptor for RANKL.     

Interleukin-18 up-regulates osteoprotegerin expression in stromal/osteoblastic cells

Osteoprotegerin (OPG) and osteoclast differentiation factor (ODF) are crucial regulators of osteoclastogenesis. To determine the biological role of interleukin (IL)-18 produced by stromal/osteoblastic cells in osteoclastogenesis, we examined the effects of IL-18 on the OPG and ODF mRNA levels in these cells. When bone marrow stromal ST2 cells, osteoblastic MC3T3-E1 cells, and mouse calvarial osteoblasts were stimulated with IL-18, the expression of OPG mRNA, but not ODF mRNA, was transiently increased, its expression reaching a maximal level at 3 h after the beginning of the culture. In accordance with this observation, all these cells expressed the mRNAs of two IL-18 receptor components and MyD88, an adapter molecule involved in IL-18 signaling. Moreover, in these cells, mitogen-activated protein kinase was phosphorylated after stimulation with IL-18. These results suggest that stromal/osteoblastic cells are IL-18-responsive cells and that IL-18 may inhibit osteoclastogenesis by up-regulating OPG expression, without stimulation of ODF production, in stromal/osteoblastic cells.