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.     

Direct inhibition of human RANK+ osteoclast precursors identifies a homeostatic function of IL-1beta

IL-1β is a key mediator of bone resorption in inflammatory settings, such as rheumatoid arthritis (RA). IL-1β promotes osteoclastogenesis by inducing RANKL expression on stromal cells and synergizing with RANKL to promote later stages of osteoclast differentiation. Because IL-1Rs share a cytosolic Toll-IL-1R domain and common intracellular signaling molecules with TLRs that can directly inhibit early steps of human osteoclast differentiation, we tested whether IL-1β also has suppressive properties on osteoclastogenesis in primary human peripheral blood monocytes and RA synovial macrophages. Early addition of IL-1β, prior to or together with RANKL, strongly inhibited human osteoclastogenesis as assessed by generation of TRAP(+) multinucleated cells. IL-1β acted directly on human osteoclast precursors (OCPs) to strongly suppress expression of RANK, of the costimulatory triggering receptor expressed on myeloid cells 2 receptor, and of the B cell linker adaptor important for transmitting RANK-induced signals. Thus, IL-1β rendered early-stage human OCPs refractory to RANK stimulation. Similar inhibitory effects of IL-1β were observed using RA synovial macrophages. One mechanism of RANK inhibition was IL-1β-induced proteolytic shedding of the M-CSF receptor c-Fms that is required for RANK expression. These results identify a homeostatic function of IL-1β in suppressing early OCPs that contrasts with its well-established role in promoting later stages of osteoclast differentiation. Thus, the rate of IL-1-driven bone destruction in inflammatory diseases, such as RA, can be restrained by its direct inhibitory effects on early OCPs to limit the extent of inflammatory osteolysis.     

Human interleukin-1-induced murine osteoclastogenesis is dependent on RANKL, but independent of TNF-alpha

Although interleukin-1 (IL-1) has been implicated in the pathogenesis of inflammatory osteolysis, the means by which it recruits osteoclasts and promotes bone destruction are largely unknown. Recently, a cytokine-driven, stromal cell-free mouse osteoclastogenesis model was established. A combination of macrophage colony stimulating factor (M-CSF) and receptor activator of NFkappaB ligand (RANKL) was proven to be sufficient in inducing differentiation of bone marrow hematopoietic precursor cells to bone-resorbing osteoclasts in the absence of stromal cells or osteoblasts. This study utilizes this model to examine the impact of human IL-1beta on in vitro osteoclastogenesis of bone marrow progenitor cells. We found that osteoclast precursor cells failed to undergo osteoclastogenesis when treated with IL-1 alone. In contrast, IL-1 dramatically up-regulated osteoclastogenesis by 2.5- to 4-folds in the presence of RANKL and M-CSF. The effect can be significantly blocked by IL-1 receptor antagonist (p < 0.01). Tumor necrosis factor-alpha (TNF-alpha) was undetectable in the culture medium of differentiating osteoclasts induced by IL-1. Adding exogenous TNF-alpha neutralizing antibody had no influence on the IL-1-induced effect as well. These results show that in the absence of stromal cells, IL-1 exacerbates osteoclastogenesis by cooperating with RANKL and M-CSF, while TNF-alpha is not involved in this IL-1-stimulated osteoclast differentiation pathway.     

Glutathione accelerates osteoclast differentiation and inflammatory bone destruction

Chronic inflammation associated with bone tissues often destructs bones, which is essentially performed by osteoclasts in the presence of immunoregulatory molecules. Hence, regulating osteoclastogenesis is crucial to develop therapeutics for bone-destructive inflammatory diseases. It is believed that reactive oxygen species (ROS) are involved in receptor activator of NF-κB (RANK) ligand (RANKL)-induced osteoclast differentiation, and, therefore, glutathione (GSH), the most abundant endogenous antioxidant, suppresses osteoclast differentiation and bone resorption by RANKL. Interestingly, GSH also contributes to inflammatory responses, and the effects of GSH on osteoclast differentiation and bone destruction under inflammatory conditions have not yet been determined. Here, we investigated how GSH affects inflammatory cytokine-stimulated osteoclast differentiation in vitro and in a mouse model of inflammatory bone destruction. We found that GSH significantly promoted TNFα-stimulated osteoclast formation, while an inhibitor of GSH synthesis, buthionine sulfoximine, suppressed it. GSH facilitated the nuclear localisation of the nuclear factor of activated T cells c1 (NFATc1) protein, a master regulator of osteoclastogenesis, as well as the expression of osteoclast marker genes in a dose-dependent manner. N-acetylcysteine, a substrate of GSH synthesis, also stimulated osteoclast formation and NFATc1 nuclear localisation. GSH did not suppress cell death after osteoclast differentiation. In mouse calvaria injected with lipopolysaccharide, GSH treatment resulted in a fivefold increase in the osteolytic lesion area. These results indicate that GSH accelerates osteoclast differentiation and inflammatory bone destruction, suggesting GSH appears to be an important molecule in the mechanisms responsible for inflammatory bone destruction by osteoclasts.     

Connection between B lymphocyte and osteoclast differentiation pathways

Osteoclasts differentiate from the hemopoietic monocyte/macrophage cell lineage in bone marrow through cell-cell interactions between osteoclast progenitors and stromal/osteoblastic cells. Here we show another osteoclast differentiation pathway closely connected with B lymphocyte differentiation. Recently the TNF family molecule osteoclast differentiation factor/receptor activator of NF-kappaB ligand (ODF/RANKL) was identified as a key membrane-associated factor regulating osteoclast differentiation. We demonstrate that B-lymphoid lineage cells are a major source of endogenous ODF/RANKL in bone marrow and support osteoclast differentiation in vitro. In addition, B-lymphoid lineage cells in earlier developmental stages may hold a potential to differentiate into osteoclasts when stimulated with M-CSF and soluble ODF/RANKL in vitro. B-lymphoid lineage cells may participate in osteoclastogenesis in two ways: they 1) express ODF/RANKL to support osteoclast differentiation, and 2) serve themselves as osteoclast progenitors. Consistent with these observations in vitro, a decrease in osteoclasts is associated with a decrease in B-lymphoid cells in klotho mutant mice (KL(-/-)), a mouse model for human aging that exhibits reduced turnover during bone metabolism, rather than a decrease in the differentiation potential of osteoclast progenitors. Taken together, B-lymphoid lineage cells may affect the pathophysiology of bone disorders through regulating osteoclastogenesis.     

alpha-Lipoic acid inhibits inflammatory bone resorption by suppressing prostaglandin E2 synthesis

alpha-Lipoic acid (LA) has been intensely investigated as a therapeutic agent for several pathological conditions, including diabetic polyneuropathy. In the present study, we examined the effects of LA on osteoclastic bone loss associated with inflammation. LA significantly inhibited IL-1-induced osteoclast formation in cocultures of mouse osteoblasts and bone marrow cells, but LA had only a marginal effect on osteoclastogenesis from bone marrow macrophages induced by receptor activator of NF-kappaB ligand (RANKL). LA inhibited both the sustained up-regulation of RANKL expression and the production of PGE2 induced by IL-1 in osteoblasts. In addition, treatment with either prostaglandin E2 (PGE2) or RANKL rescued IL-1-induced osteoclast formation inhibited by LA or NS398, a specific cyclooxygenase-2 (COX-2) inhibitor, in cocultures. LA blocked IL-1-induced PGE2 production even in the presence of arachidonic acid, without affecting the expression of COX-2 and membrane-bound PGE2 synthase. Dihydrolipoic acid (the reduced form of LA), but not LA, attenuated recombinant COX-2 activity in vitro. LA also inhibited osteoclast formation and bone loss induced by IL-1 and LPS in mice. Our results suggest that the reduced form of LA inhibits COX-2 activity, PGE2 production, and sustained RANKL expression, thereby inhibiting osteoclast formation and bone loss in inflammatory conditions.     

TNF‐α has both stimulatory and inhibitory effects on mouse monocyte‐derived osteoclastogenesis

Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF‐α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31^hiLy‐6C^?), myeloid blasts (CD31⁺Ly‐6C⁺), and monocytes (CD31^?Ly‐6C^hi) were sorted from mouse bone marrow using flow cytometry and cultured with M‐CSF and RANKL, with or without TNF‐α. Surprisingly, TNF‐α prevented the differentiation of TRAcP⁺ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL‐1β or IL‐6. When monocytes were pre‐cultured with M‐CSF and RANKL followed by exposure to TNF‐α, a stimulatory effect was found. TNF‐α also stimulated monocytes’ osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF‐α was added to monocytes cultured on plastic, RANK, NFATc1, and TRAcP were significantly down‐regulated while TNF‐αR1 and TNF‐αR2 were up‐regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF‐α, indicating an altered ratio of bound‐RANK/unbound‐RANK. Our findings suggest a diverse role of TNF‐α on monocytes’ osteoclastogenesis: it affects the RANK‐signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre‐cultured with M‐CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte‐derived osteoclast formation away from the bone.

     

Osteoprotegerin and inflammation

RANK, RANKL, and OPG have well established regulatory effects on bone metabolism. RANK is expressed at very high levels on osteoclastic precursors and on mature osteoclasts, and is required for differentiation and activation of the osteoclast. The ligand, RANKL binds to its receptor RANK to induce bone resorption. RANKL is a transmembrane protein expressed in various cells type and particularly on osteoblast and activated T cells. RANKL can be cleaved and the soluble form is active. Osteoprotegerin decoy receptor (OPG), a member of the TNF receptor family expressed by osteoblasts, strongly inhibits bone resorption by binding with high affinity to its ligand RANKL, thereby preventing RANKL from engaging its receptor RANK. This system is regulated by the calciotropic hormones. Conversely, the effects of RANKL, RANK, and OPG on inflammatory processes, most notably on the bone resorption associated with inflammation, remain to be defined. The RANK system seems to play a major role in modulating the immune system. Activated T cells express RANKL messenger RNA, and knock-out mice for RANKL acquire severe immunological abnormalities and osteopetrosis. RANKL secretion by activated T cells can induce osteoclastogenesis. These mechanisms are enhanced by cytokines such as TNF-alpha, IL-1, and IL-17, which promote both inflammation and bone resorption. Conversely, this system is blocked by OPG, IL-4, and IL-10, which inhibit both inflammation and osteoclastogenesis. These data may explain part of the abnormal phenomena in diseases such as rheumatoid arthritis characterized by both inflammation and destruction. Activated T cells within the rheumatoid synovium express RANKL. Synovial cells are capable of differentiating to osteoclast-like cells under some conditions, including culturing with M-CSF and RANKL. This suggests that the bone erosion seen in rheumatoid arthritis may result from RANKL/RANK system activation by activated T cells. This opens up the possibility that OPG may have therapeutic effects mediated by blockade of the RANKL/RANK system.     

Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo

Wear particle‐stimulated inflammatory bone destruction and the consequent aseptic loosening remain the primary causes of artificial prosthesis failure and revision. Previous studies have demonstrated that curcumin has a protective effect on bone disorders and inflammatory diseases and can ameliorate polymethylmethacrylate‐induced osteolysis in vivo. However, the effect on immunomodulation and the definitive mechanism by which curcumin reduces the receptor activators of nuclear factor‐kappa B ligand (RANKL)‐stimulated osteoclast formation and prevents the activation of osteoclastic signalling pathways are unclear. In this work, the immunomodulation effect and anti‐osteoclastogenesis capacities exerted by curcumin on titanium nanoparticle‐stimulated macrophage polarization and on RANKL‐mediated osteoclast activation and differentiation in osteoclastic precursor cells in vitro were investigated. As expected, curcumin inhibited RANKL‐stimulated osteoclast maturation and formation and had an immunomodulatory effect on macrophage polarization in vitro. Furthermore, studies aimed to identify the potential molecular and cellular mechanisms revealed that this protective effect of curcumin on osteoclastogenesis occurred through the amelioration of the activation of Akt/NF‐κB/NFATc1 pathways. Additionally, an in vivo mouse calvarial bone destruction model further confirmed that curcumin ameliorated the severity of titanium nanoparticle‐stimulated bone loss and destruction. Our results conclusively indicated that curcumin, a major biologic component of Curcuma longa with anti‐inflammatory and immunomodulatory properties, may serve as a potential therapeutic agent for osteoclastic diseases.     

Antioxidant alpha-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-kappaB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-kappaB ligand and tumor necrosis factor-alpha

The relationship between oxidative stress and bone mineral density or osteoporosis has recently been reported. As bone loss occurring in osteoporosis and inflammatory diseases is primarily due to increases in osteoclast number, reactive oxygen species (ROS) may be relevant to osteoclast differentiation, which requires receptor activator of nuclear factor-kappaB ligand (RANKL). Tumor necrosis factor-alpha (TNF-alpha) frequently present in inflammatory conditions has a profound synergy with RANKL in osteoclastogenesis. In this study, we investigated the effects of alpha-lipoic acid (alpha-LA), a strong antioxidant clinically used for some time, on osteoclast differentiation and bone resorption. At concentrations showing no growth inhibition, alpha-LA potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven either by a high-dose RANKL alone or by a low-dose RANKL plus TNF-alpha (RANKL/TNF-alpha). alpha-LA abolished ROS elevation by RANKL or RANKL/TNF-alpha and inhibited NF-kappaB activation in osteoclast precursor cells. Specifically, alpha-LA reduced DNA binding of NF-kappaB but did not inhibit IKK activation. Furthermore, alpha-LA greatly suppressed in vivo bone loss induced by RANKL or TNF-alpha in a calvarial remodeling model. Therefore, our data provide evidence that ROS plays an important role in osteoclast differentiation through NF-kappaB regulation and the antioxidant alpha-lipoic acid has a therapeutic potential for bone erosive diseases.     

Exposure to receptor-activator of NFkappaB ligand renders pre-osteoclasts resistant to IFN-gamma by inducing terminal differentiation

While it has been established that IFN-gamma is a strong activator of macrophages and a potent inhibitor of osteoclastogenesis in vitro, it is also known that this cytokine is produced in particular settings of inflammatory bone loss, such as infection and psoriatic arthritis. Because of the different kinetics between rapid IFN-gamma macrophage activation (<24 hours) and the slower receptor-activator of NFkappaB ligand (RANKL) osteoclast differentiation (7 days), we postulated that IFN-gamma would have different effects on early-stage and late-stage osteoclast precursors. In RAW264.7 cells and primary splenocyte cultures, pretreatment with RANKL rendered these cells resistant to maximally anti-osteoclastogenic doses of IFN-gamma. These cells were also resistant to IFN-gamma-induced nitric oxide production, morphological change, and surface upregulation of CD11b and receptor-activator of NFkappaB, suggesting that early exposure of osteoclast precursors to RANKL induces a broad resistance to the cellular effects of IFN-gamma. Changes in STAT1 activation did not correlate with this resistance, as IFN-gamma activated STAT1 equally in both early-stage and late-stage pre-osteoclasts. Furthermore, we failed to observe changes in TRAF6 expression following IFN-gamma treatment in pre-osteoclasts. Together these data support a model of inflammatory bone loss in which early exposure to RANKL can prime osteoclast precursors to form in the presence of high levels of IFN-gamma using mechanisms independent of the signal molecules STAT1 and TRAF6.     

Stachydrine prevents LPS‐induced bone loss by inhibiting osteoclastogenesis via NF‐κB and Akt signalling

Osteoclast overactivation‐induced imbalance in bone remodelling leads to pathological bone destruction, which is a characteristic of many osteolytic diseases such as rheumatoid arthritis, osteoporosis, periprosthetic osteolysis and periodontitis. Natural compounds that suppress osteoclast formation and function have therapeutic potential for treating these diseases. Stachydrine (STA) is a bioactive alkaloid isolated from Leonurus heterophyllus Sweet and possesses antioxidant, anti‐inflammatory, anticancer and cardioprotective properties. However, its effects on osteoclast formation and function have been rarely described. In the present study, we found that STA suppressed receptor activator of nuclear factor‐κB (NF‐κB) ligand (RANKL)‐induced osteoclast formation and bone resorption, and reduced osteoclast‐related gene expression in vitro. Mechanistically, STA inhibited RANKL‐induced activation of NF‐κB and Akt signalling, thus suppressing nuclear factor of activated T cells c1 induction and nuclear translocation. In addition, STA alleviated bone loss and reduced osteoclast number in a murine model of LPS‐induced inflammatory bone loss. STA also inhibited the activities of NF‐κB and NFATc1 in vivo. Together, these results suggest that STA effectively inhibits osteoclastogenesis both in vitro and in vivo and therefore is a potential option for treating osteoclast‐related diseases.     

RANKL-stimulated TNFalpha production in osteoclast precursor cells promotes osteoclastogenesis by modulating RANK signaling pathways

Although TNFalpha is known to be an important factor for bone resorption, particularly in inflammatory bone diseases, the relevance between RANKL and TNFalpha in osteoclastogenesis remains unclear. In this study we examined the mechanism of TNFalpha induced osteoclastogenesis and its downstream signaling. We show that osteoclastogenesis is suppressed by anti-TNFalpha- and anti-TNF receptor type I (TNFRI)-antibodies and in TNFalpha- and TNFRI-deficient mice using in vitro culture systems: (1) co-culture of mouse spleen derived osteoclast precursor cells (pOCs) with osteoblasts, (2) pure pOC culture and (3) RAW264.7 cells in presence of RANKL. Furthermore, TNFalpha production in pOCs was stimulated by RANKL. Endogenous TNFalpha in pOCs induced c-Fos and NFATc1. Expression rates of NFATc1 and c-Fos were significantly decreased in TNFalpha- and TNFRI-deficient pOCs during osteoclastogenesis. These results indicate that TNFalpha is induced by RANKL in pOCs and serves as an autocrine factor promoting osteoclastogenesis through c-Fos and NFATc1 activation.     

IL-23 induces receptor activator of NF-kappaB ligand expression on CD4+ T cells and promotes osteoclastogenesis in an autoimmune arthritis model

IL-23, a clinically novel cytokine, targets CD4(+) T cells. Recent IL-1Ra(-/-) mouse studies have demonstrated that IL-23 indirectly stimulates the differentiation of osteoclast precursors by enhancing IL-17 release from CD4(+) T cells. IL-17, in turn, stimulates osteoclastogenesis in osteoclast precursor cells. In this study, we found that IL-23 up-regulates receptor activator of NF-kappaB ligand expression by CD4(+) T cells, and thus contributes to osteoclastogenesis. This indirect pathway is mediated by NF-kappaB and STAT3. We have also demonstrated that IL-23 can influence osteoclastogenesis positively under the special conditions in the IL-1-dominant milieu of IL-1Ra(-/-) mice. We propose that IL-23-enhanced osteoclastogenesis is mediated mainly by CD4(+) T cells. The results of this study show that IL-23 is a promising therapeutic target for the treatment of arthritis-associated bone destruction.     

Tumor necrosis factor-alpha mediates RANK ligand stimulation of osteoclast differentiation by an autocrine mechanism

Osteoblasts or bone marrow stromal cells are required as supporting cells for the in vitro differentiation of osteoclasts from their progenitor cells. Soluble receptor activator of nuclear factor-kappaB ligand (RANKL) in the presence of macrophage colony-stimulating factor (M-CSF) is capable of replacing the supporting cells in promoting osteoclastogenesis. In the present study, using Balb/c-derived cultures, osteoclast formation in both systems-osteoblast/bone-marrow cell co-cultures and in RANKL-induced osteoclastogenesis-was inhibited by antibody to tumor necrosis factor-alpha (TNF-alpha), and was enhanced by the addition of this cytokine. TNF-alpha itself promoted osteoclastogenesis in the presence of M-CSF. However, even at high concentrations of TNF-alpha the efficiency of this activity was much lower than the osteoclastogenic activity of RANKL. RANKL increased the level of TNF-alpha mRNA and induced TNF-alpha release from osteoclast progenitors. Furthermore, antibody to p55 TNF-alpha receptors (TNF receptors-1) (but not to p75 TNF-alpha receptors (TNF receptors-2) inhibited effectively RANKL- (and TNF-alpha() induced osteoclastogenesis. Anti-TNF receptors-1 antibody failed to inhibit osteoclastogenesis in C57BL/6-derived cultures. Taken together, our data support the hypothesis that in Balb/c, but not in C57BL/6 (strains known to differ in inflammatory responses and cytokine modulation), TNF-alpha is an autocrine factor in osteoclasts, promoting their differentiation, and mediates, at least in part, RANKL's induction of osteoclastogenesis.