Oskar Hoffmann

JNK1 modulates osteoclastogenesis through both c-Jun phosphorylation-dependent and -independent mechanisms.

Phosphorylation of the N-terminal domain of Jun by the Jun kinases (JNKs) modulates the transcriptional activity of AP-1, a dimeric transcription factor typically composed of c-Jun and c-Fos, the latter being essential for osteoclast differentiation. Using mice lacking JNK1 or JNK2, we demonstrate that JNK1, but not JNK2, is specifically activated by the osteoclast-differentiating factor RANKL. Activation of JNK1, but not JNK2, is required for efficient osteoclastogenesis from bone marrow monocytes (BMMs). JNK1 protects BMMs from RANKL-induced apoptosis during differentiation. In addition, BMMs from mice carrying a mutant of c-Jun phosphorylation sites (JunAA/JunAA), as well as cells lacking either c-Jun or JunD, which is another JNK substrate, revealed that c-Jun phosphorylation and c-Jun itself, but not JunD, are essential for efficient osteoclastogenesis. Moreover, JNK1-dependent c-Jun phosphorylation in response to RANKL is not involved in the anti-apoptotic function of JNK1. Thus, these data provide genetic evidence that JNK1 activation modulates osteoclastogenesis through both c-Jun-phosphorylation-dependent and -independent mechanisms.

Estrogen-dependent and C-C chemokine receptor-2-dependent pathways determine osteoclast behavior in osteoporosis

Understanding the mechanisms of osteoclastogenesis is crucial for developing new drugs to treat diseases associated with bone loss, such as osteoporosis. Here we report that the C-C chemokine receptor-2 (CCR2) is crucially involved in balancing bone mass. CCR2-knockout mice have high bone mass owing to a decrease in number, size and function of osteoclasts. In normal mice, activation of CCR2 in osteoclast progenitor cells results in both nuclear factor-κB (NF-κB) and extracellular signal–related kinase 1 and 2 (ERK1/2) signaling but not that of p38 mitogen-activated protein kinase or c-Jun N-terminal kinase. The induction of NF-κB and ERK1/2 signaling in turn leads to increased surface expression of receptor activator of NF-κB (RANK, encoded by Tnfrsf11a), making the progenitor cells more susceptible to RANK ligand-induced osteoclastogenesis. In ovariectomized mice, a model of postmenopausal osteoporosis, CCR2 is upregulated on wild-type preosteoclasts, thus increasing the surface expression of RANK on these cells and their osteoclastogenic potential, whereas CCR2-knockout mice are resistant to ovariectomy-induced bone loss. These data reveal a previously undescribed pathway by which RANK, osteoclasts and bone homeostasis are regulated in health and disease.

Heme oxygenase 1 (HO-1) regulates osteoclastogenesis and bone resorption

Heme oxygenase 1 (HO‐1) plays an important role in vascular disease, transplantation, and inflammation. In animal models of acute and chronic inflammation, induction of HO‐1 has anti‐inflammatory and cytoprotective properties. Since inflammation is an important trigger of osteoclastogenesis, we hypothesized that HO‐1 might influence osteoclastogenesis. We investigated the effects of induction of HO‐1 on osteoclast formation in vitro and in vivo. Furthermore, we addressed the role of HO‐1 in inflammatory bone loss in humans. When HO‐1 was induced by hemin in vitro, a significant dose‐dependent inhibition of osteoclastogenesis was observed. Up‐regulation of HO‐1 was mediated by activation of MAPK and primarily prevented differentiation of osteoclast precursors to osteoclasts, whereas it did not affect mature osteoclasts. Anti‐osteoclastogenic properties of hemin were based on a down‐regulation of c‐fms, RANK, TRAF‐6, and c‐fos. In addition, induction of HO‐1 inhibited TNF‐triggered osteoclast differentiation in vitro as well as LPS‐driven inflammatory bone loss in vivo. Furthermore, HO‐1 induction suppressed osteoclastogenesis and bone destruction in a TNF‐mediated arthritis. In line, assessment of synovial tissue from rheumatoid arthritis patients revealed that osteoclasts are usually HO‐1 negative. Moreover, serum levels of bilirubin, a metabolite of HO‐1, were elevated in rheumatoid arthritis patients without bone damage, suggesting HO‐1 affects bone loss in humans. In summary, these data indicate that HO‐1 negatively regulates osteoclastogenesis, leading to a positive net balance of bone.

Essential role of RSK2 in c-Fos–dependent osteosarcoma development

Inactivation of the growth factor-regulated S6 kinase RSK2 causes Coffin-Lowry syndrome in humans, an X-linked mental retardation condition associated with progressive skeletal abnormalities. Here we show that mice lacking RSK2 develop a progressive skeletal disease, osteopenia due to impaired osteoblast function and normal osteoclast differentiation. The phenotype is associated with decreased expression of Phex, an endopeptidase regulating bone mineralization. This defect is probably not mediated by RSK2-dependent phosphorylation of c-Fos on serine 362 in the C-terminus. However, in the absence of RSK2, c-Fos-dependent osteosarcoma formation is impaired. The lack of c-Fos phosphorylation leads to reduced c-Fos protein levels, which are thought to be responsible for decreased proliferation and increased apoptosis of transformed osteoblasts. Therefore, RSK2-dependent stabilization of c-Fos is essential for osteosarcoma formation in mice and may also be important for human osteosarcomas.

CD44 is a determinant of inflammatory bone loss

Chronic inflammation is a major trigger of local and systemic bone loss. Disintegration of cell–matrix interaction is a prerequisite for the invasion of inflammatory tissue into bone. CD44 is a type I transmembrane glycoprotein that connects a variety of extracellular matrix proteins to the cell surface. Tumor necrosis factor (TNF) is a major inducer of chronic inflammation and its overexpression leads to chronic inflammatory arthritis. By generating CD44−/− human TNF-transgenic (hTNFtg) mice, we show that destruction of joints and progressive crippling is far more severe in hTNFtg mice lacking CD44, which also develop severe generalized osteopenia. Mutant mice exhibit an increased bone resorption due to enhanced number, size, and resorptive capacity of osteoclasts, whereas bone formation and osteoblast differentiation are not affected. Responsiveness of CD44-deficient osteoclasts toward TNF is enhanced and associated with increased activation of the p38 mitogen-activated protein kinase. These data identify CD44 as a critical inhibitor of TNF-driven joint destruction and inflammatory bone loss.

Gamma interferon inhibits basal and interleukin 1-induced prostaglandin production and bone resorption in neonatal mouse calvaria

Production of the osteolytic arachidonic acid metabolites, prostaglandin (PG) E2, PGI2 and PGF2 alpha, by neonatal mouse calvariae was quantitated by gas chromatography/mass spectrometry. Mouse recombinant interleukin 1 (rIL-1) raised medium levels of PGE2 and PGI2 (measured as 6-keto-PGF1 alpha) in the dose range tested (1.0-10.0 U/ml culture medium), while an effect on PGF2 was only observed at 10 U/ml. Bone resorption in response to rIL-1 reached a plateau at 3.0 U/ml. Mouse recombinant gamma-interferon (rIFN-gamma) between 100-500 U/ml suppressed basal PG synthesis and spontaneous resorption of cultured bone. In addition, IFN-gamma at 100 U/ml prevented stimulation of PG synthesis by 3.0 U/ml rIL-1 and thereby reduced the bone resorbing activity of the cytokine by at least 60%. 5 X 10(-7) M indomethacin was equally effective in suppression of PG synthesis and bone resorption. The present study provides evidence that IFN-gamma inhibits PG synthesis and consequently resorption of cultured bone.

Recombinant γ-interferon inhibits prostaglandin-mediated and parathyroid hormone-induced bone resorption in cultured neonatal mouse calvaria

γ‐Interferon Bone resorption Neonatal mouse calvaria Prostaglandin synthesis Parathyroid hormone Calcitonin

Interaction of triiodothyronine with 1α, 25-dihydroxyvitamin D3 on interleukin-6-dependent osteoclast-like cell formation in mouse bone marrow cell cultures

In mouse bone marrow cultures, the formation of osteoclast-like, that is, tartrate-resistant acid phosphatase-positive (TRAP+) and calcitonin (CT) receptor-positive multinucleated cells (MNCs), induced by 10(-10) to 10(-8) mol/L 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], could be augmented by triiodothyronine (T3), which alone had no effect on osteoclast-like cell formation. The permissive effect of T3 increased the response to 1alpha,25(OH)2D3 by approximately one order of magnitude. Linear concentration dependence was observed between 10(-11) and 10(-8) mol/L T3. Importantly, inhibition of prostaglandin synthesis by indomethacin significantly impeded osteoclast-like cell formation by 1alpha,25(OH)2D3 and abrogated the effect of T3 thereon. Basal interleukin-6 (IL-6) production by cultured marrow cells was significantly stimulated by 1alpha,25(OH)2D3. However, even at an exceedingly high concentration of 20 ng/mL, IL-6 was ineffective in inducing osteoclast-like cell formation. Therefore, any hormonally induced rise in IL-6 release from bone marrow cells could not account for the observed changes in TRAP+ MNC numbers. Nevertheless, the stimulatory effect of 1alpha,25(OH)2D3 on osteoclastogenesis was partially dependent on IL-6 because it could be significantly blocked by a neutralizing monoclonal anti-IL-6 antibody, and to the same extent by a monoclonal anti-IL-6 receptor antibody. Unimpaired signaling through the IL-6/IL-6R system is also a prerequisite for the auxiliary effect of T3 on induction of osteoclast-like cells by 1alpha,25(OH)2D3. Our data provide evidence that 1alpha,25(OH)2D3 induces osteoclast-like cell formation, at least in part, in an IL-6-dependent mode of action, which is also subject to modulation by T3. The mechanism of interaction of the two hormones apparently involves joint stimulation of prostaglandin synthesis.

Removal of the surface layers of human cortical bone allografts restores in vitro osteoclast function reduced by processing and frozen storage

A major complication of cortical bone grafting is nonunion at the host-graft junction. Many factors are thought to be involved in successful engraftment including the quality of the graft and the host response to it. In particular, the recipient osteoclasts (OCs) play a critical role by resorbing the engrafted bone. Thus, effective engraftment may depend on the inherent biological properties of the bone graft, which subsequently correlates with early and effective OC resorption. Normally, bone grafts are stored and processed by freezing, freeze-drying, irradiation, and lipid extraction. We sought to determine whether processing and storage affected bone quality, as evaluated by OC bone resorption. Cortical bone specimens from six human donors were either fresh, frozen at -75 degrees C, or had undergone combinations of freezing at -75 degrees C, freeze-drying, lipid extraction, irradiation, and treatment with hydrogen peroxide. Bone slices of 0.5-mm thickness taken from the surface, beneath the surface, and at a depth of 7.5 mm were incubated with isolated rabbit OCs and resorption lacunae were measured. We observed highest OC activity with fresh bone followed by frozen, partially processed, and fully processed bone. When allografts were stored at -75 degrees C for 12 months, there was up to a 4.2-fold reduction in OC activity on the surface layer. Additionally, we found reduced OC activity upon the outer surface bone compared to the inner layers. Removal of more than 0.5 mm of frozen and processed bone significantly improved OC activity. These results imply that inner bone layers of stored and processed bone allografts are protected against degradation of bone matrix components, except when frozen for extended periods of time. Taken together, these data suggest that bone allografts should be stored for less than 1 year and require the removal of at least 0.5 mm from their surface prior to transplantation.

Comparison between the effects of forskolin and calcitonin on bone resorption and osteoclast morphology in vitro.

The adenylate cyclase activator forskolin (1-10 mumol/L) inhibited 45Ca release from parathyroid hormone (PTH; 10 nmol/L) stimulated prelabeled neonatal mouse calvaria in short term culture (24 h). This effect of forskolin was potentiated by rolipram, Ro 20-1724, and isobutyl-methylxanthine, three structurally different inhibitors of cyclic AMP phosphodiesterase. Forskolin (10 mumol/L) and calcitonin (30 mU/mL) inhibited the mobilization of stable calcium and inorganic phosphate as well as the release of the lysomal enzymes beta-glucuronidase and beta-N-acetylglucosaminidase from PTH-stimulated unlabeled bones. Osteoclasts in PTH-stimulated calvaria showed active ruffled borders with numerous membrane infoldings. Treatment of PTH-stimulated bones with forskolin and calcitonin resulted in a rapid (2 h) loss of the active ruffled border. In addition, forskolin and calcitonin induced similar changes with respect to the number and size distribution of cytoplasmic vesicles in PTH-activated osteoclasts. After 24 h, all signs of osteoclast inactivation were still prominent, whereas after 48 h of treatment with forskolin or calcitonin, the reappearance of a ruffled border on a number of osteoclasts signaled an escape from the inhibitory action of both calcitonin or forskolin. These data indicate that forskolin inhibits bone resorption by a cyclic AMP dependent mechanism and that the effect of forskolin and calcitonin on bone resorption and osteoclast morphology are comparable. These observations lend further support to the view that cyclic AMP may be an intracellular mediator of the inhibitory action of calcitonin on multinucleated osteoclasts.