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Bone undergoes continuous remodeling due to balanced bone formation and resorption mediated by osteoblasts and osteoclasts, respectively. Osteoclasts arise from the macrophage lineage, and their differentiation is dependent on RANKL, a member of the TNF family of cytokines. Here, we have provided evidence that RANKL controls the expression of 3BP2, an adapter protein that is required for activation of SRC tyrosine kinase and simultaneously coordinates the attenuation of [beta]-Catenin, both of which are required to execute the osteoclast developmental program. We found that RANKL represses the transcription of the E3 ubiquitin ligase RNF146 through an NF-[kappa]B-related inhibitory element in the RNF146 promoter. RANKL-mediated suppression of RNF146 results in the stabilization of its substrates, 3BP2 and AXIN1, which consequently triggers the activation of SRC and attenuates the expression of [beta]-Catenin, respectively. Depletion of RNF146 caused hypersensitivity to LPS-induced TNF-[alpha] production in vivo. RNF146 thus acts as an inhibitory switch to control osteoclastogenesis and cytokine production and may be a control point underlying the pathogenesis of chronic inflammatory diseases., Introduction RANKL, a member of the TNF superfamily of cytokines, is an obligate factor required for osteoclast differentiation and survival, lymph node organogenesis, lymphocyte development, and breast epithelial proliferation (1-6). [...]

Vascular calcification is a cardiovascular disease divided into two distinct areas: intimal and medial. Intimal calcification involves plaque build-up in the arteries, which in turn leads to heart attack and stroke. Medial calcification is result of mineral deposition in the interior walls of the arteries. This results in complications including hypertension, heart attack, and stroke. Interestingly, research has shown that medial vascular calcification is physiologically similar to bone development in the body. Bone remodeling is a process that occurs continually in the body. This process keeps bone mass consistent through the continual resorbing of bone by osteoclasts and deposition of bone by osteoblasts. Smooth muscle cells in the artery walls are differentiating into osteoblast-like cells and depositing mineral inside the artery walls. Evidence has shown that osteoclast cell therapy is a potential pathway for treatment of vascular calcification. In this therapy, osteoclasts would resorb mineral the same way bone remodeling is done in vivo. By introducing osteoclasts to sites of calcification, it is hypothesized that mineralization can be resorbed. To introduce osteoclast-like cells to sites of calcification, monocyte differentiation is a potential option. Monocytes are mobile inflammatory response cells normally found in bone and are also commonly found at sites of high calcification. Additionally, monocytes have the capacity to differentiate into osteoclast-like cells under the right conditions. Our objective is to take advantage of this by demineralizing vascular calcification through differentiation of monocytes to osteoclast-like cells in vitro. Keywords: Vascular Calcification, Osteoclasts, Demineralization, Cardiovascular, monocytes, arteriosclerosis., INTRODUCTION Cardiovascular disease is the leading cause of death in the United States. According to the Center for Disease Control and Prevention, about 600,000 people succumb due to heart related [...]

Vascular calcification is the deposition of calcium mineral in blood vessels and can cause increased vascular stiffness, resulting in an increased risk for cardiovascular complications. It has recently been determined that calcification resembles the active process of bone formation, with smooth muscle cells being able to differentiate into bone-forming osteoblast-like cells. Another key characteristic of vascular calcification is the breakdown of elastin, the protein responsible for providing blood vessels their elasticity. It is believed that degraded elastin could provide sites for the initiation of vascular calcification. A recent study has shown that bone-absorbing cells, osteoclasts, can demineralize calcified elastin. To improve upon this study, degraded elastin-targeted nanoparticles will be used to target sites of calcification and cause resident macrophages, inflammatory cells believed to be involved in vascular calcification, to differentiate into osteoclasts. Iron oxide nanoparticles, which have already been used in the imaging of macrophage-rich areas in arteries with magnetic resonance imaging (MRI), will be modified with anti-tropoelastin antibodies. Once injected, the nanoparticles will target sites of calcification, acting as a contrast agent for MRI and delivering 1,25 dihydroxyvitamin D3 and macrophage colony-stimulating factor to the nearby macrophages and circulating monocytes to induce osteoclastic differentiation and facilitate demineralization of the calcified arteries. With an estimated 18 million people dying from cardiovascular diseases world wild, research in this field is essential, and this proposed research could be pivotal in the understanding, treatment, and diagnosis of vascular calcification. Keywords: vascular calcification, degraded elastin iron oxide nanoparticles, osteoclast demineralization, INTRODUCTION Arterial calcification is the calcification of blood vessels, which occur in both the inner (intimal) and middle (medial) layers, and is associated with atherosclerosis, diabetes, and chronic kidney disease [...]

The detection of estrogen receptor-α (ERα) in osteoblasts and osteoclasts over 20 years ago suggested that direct effects of estrogens on both of these cell types are responsible for their [...]

Osteoarthritis and lesions to cartilage tissue are diseases that frequently result in impaired joint function and patient disability. The treatment of osteoarthritis, along with local bone defects and systemic skeletal diseases, remains a significant clinical challenge for orthopaedic surgeons. Several bone morphogenetic proteins (BMPs) are known to have osteoinductive effects, whereof BMP-2 and BMP-7 are already approved for clinical applications. There is growing evidence that the metabolism of bone as well as the cartilage damage associated with the above disease processes are strongly inter-related with the interactions of the inflammation-related pathways (in particular prostaglandin [E.sub.2] ([PGE.sub.2])) and osteogenesis (in particular bone morphogenetic protein-2 (BMP-2)). There is strong evidence that the pathways of prostaglandins and bone morphogenetic proteins are intertwined, and they have recently come into focus in several experimental and clinical studies. This paper focuses on [PGE.sub.2] and BMP-2 intertwining pathways in bone and cartilage metabolism, and summarizes the recent experimental and clinical data. Key words: osteoblasts, osteoclasts, prostaglandins, bone morphogenetic proteins, cellular signaling. L'osteoarthrite et les lesions du cartilage sont des maladies qui resultent frequemment en une reduction de la fonction articulaire et en une invalidite du patient. Le traitement de l'osteoarthrite qui s'accompagne d'anomalies osseuses locales et de maladies squelettiques systemiques pose toujours un defi clinique significatif aux chirurgiens orthopediques. Plusieurs proteines morphogenes de l'os, les BMP, sont connues pour exercer des effets osteoinducteurs, parmi lesquelles BMP-2 et BMP-6 sont deja approuvees aux fins d'applications cliniques. Il y a de plus en plus de preuves que le metabolisme de l'os et le dommage au cartilage associe aux maladies mentionnees plus haut sont fortement interrelies aux interactions des voies de l'inflammation (notamment la voie de la prostaglandine [E.sub.2], [PGE.sub.2]) et de l'osteogenese (notamment la voie de la bone morphogenetic protein-2, BMP-2). Il existe une preuve solide que les voies des prostaglandines et des BMP s'entrecroisent, et elles ont ete l'objet d'une attention particuliere dans plusieurs etudes experimentales et cliniques. Cet article se concentre sur l'entrecroisement des voies de la [PGE.sub.2] et de la BMP-2 dans le metabolisme de l'os et du cartilage, et resume les dernieres donnees experimentales et cliniques. Mots-cles : osteoblastes, osteoclastes, prostaglandines, proteines morphogenes de l'os, signalisation cellulaire. [Traduit par la Redaction], Relevance of prostaglandins and bone morphogenetic proteins in orthopaedics Prostaglandins (PG) play an important role in bone formation, inflammation processes, and perfusion. They are lipid, arachidonic acid-derived mediators that are [...]

Bone metabolism results from a balance between osteoclast-driven bone resorption and osteoblast-mediated bone formation. Diseases such as periodontitis and rheumatoid arthritis are characterized by increased bone destruction due to enhanced osteoclastogenesis (1,2). Here we report that interferon regulatory factor-8 (IRF-8), a transcription factor expressed in immune cells, is a key regulatory molecule for osteoclastogenesis. IRF-8 expression in osteoclast precursors was downregulated during the initial phase of osteoclast differentiation induced by receptor activator of nuclear factor-[kappa]]B ligand (RANKL), which is encoded by the Tnfsf11 gene. Mice deficient in Irf8 showed severe osteoporosis, owing to increased numbers of osteoclasts, and also showed enhanced bone destruction after lipopolysaccharide (LPS) administration. [Irf8.sup.-/-] osteoclast precursors underwent increased osteoclastogenesis in response to RANKL and tumor necrosis factor-[alpha] (TNF-[alpha]). IRF-8 suppressed osteoclastogenesis by inhibiting the function and expression of nuclear factor of activated T cells c1 (NFATc1). Our results show that IRF-8 inhibits osteoclast formation under physiological and pathological conditions and suggest a model where downregulation of inhibitory factors such as IRF-8 contributes to RANKL-mediated osteoclastogenesis., Osteoclasts are multinucleated giant cells derived from the monocyte and macrophage lineage. Their differentiation is triggered by RANKL in the presence of macrophage colony-stimulating factor (M-CSF, encoded by the Csf1 [...]

The aim of the present study was to evaluate the effect of dietary lactoferrin on bone metabolism in vivo using a postmenopausal animal model. We investigated whether bovine lactoferrin (bLF) ingestion could prevent bone loss in ovariectomized mice. Twelve-week-old female C3H mice either ovariectomized or sham operated were fed for 27 wk with the control diet (AIN-93M with 140 g of total milk protein as a protein source per kg of diet). Four groups of ovariectomized mice received diets including different concentrations of bLF (1, 5, 10, or 20 g of total milk protein were replaced by bLF). Ovariectomy induced a decreased uterine weight and a smaller gain of bone mineral density. Immunoreactive bLF was detected in the peripheral blood, and its concentration was related to the amount of bLF ingestion, bLF supplementation to the diet improved bone mineral density (BMD) and femoral failure load in a dose-dependent manner. We confirmed the direct effects of bLF in vitro using established and primary cultures of murine bone cells. Addition of bLF to the culture medium at a concentration of between 1 and 1,000 [micro]g/ml stimulated both cell growth and differentiation of osteoblastic MC3T3 cells while inhibiting the growth of preosteoclastic RAW 267.4 cells. In primary culture of mixed bone cells, an enhanced osteoblast differentiation was associated with an inhibition of osteoclast differentiation at lower bLF concentrations (1-10 [micro]g/ml). In conclusion, these findings suggest that dietary lactoferrin supplementation can have a beneficial effect on postmenopausal bone loss by modulating bone formation and resorption. osteoblast; osteoclast; bone mineral density

Regulation of the formation and function of bone-resorbing osteoclasts (OCs) is a key to understanding the pathogenesis of skeletal disorders. Gene-targeting studies have shown that the RANK signaling pathway plays a critical role in OC differentiation and function. Although pharmaceutical blockade of RANK may be a viable strategy for preventing bone destruction, RANK is implicated in multiple biological processes. Recently, a cytoplasmic motif of RANK was identified that may be specifically involved in OC differentiation. Here, we developed a cell-permeable inhibitor termed the RANK receptor inhibitor (RRI), which targets this motif. The RRI peptide blocked RANKL-induced OC formation from murine bone marrow--derived macrophages. Furthermore, RRI inhibited the resorptive function of OCs and induced OC apoptosis. Treatment with the peptide impaired downstream signaling of RANK linked to Vav3, Rac1, and Cdc42 and resulted in disruptions of the actin cytoskeleton in differentiated OCs. In addition, RRI blocked inflammation-induced bone destruction and protected against ovariectomy-induced bone loss in mice. These data may be useful in the development of selective therapeutic agents for the treatment of osteoporosis and other bone diseases., Introduction Osteoclasts (OCs) are large multinucleated cells (MNCs) that are unique in their ability to resorb bone. Although their activity is normally integrated with the requirements of skeletal morphogenesis and [...]

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-[kappa]B (NF-[kappa]B) 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-[kappa]B and ERK1/2 signaling in turn leads to increased surface expression of receptor activator of NF-[kappa]B (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., Osteoporosis is a major health problem particularly affecting the aging population. In industrialized societies, it is an endemic disease characterized by an imbalance in skeletal turnover (1). This leads to [...]

Osteoclasts are multinucleated cells that resorb bone. Although osteoclasts originate from the monocyte/macrophage lineage, osteoclast precursors are not well characterized in vivo. The relationship between proliferation and differentiation of osteoclast precursors is examined in this study using murine macrophage cultures treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-[kappa]B (RANK) ligand (RANKL). Cell cycle-arrested quiescent osteoclast precursors (QuOPs) were identified as the committed osteoclast precursors in vitro. In vivo experiments show that QuOPs survive for several weeks and differentiate into osteoclasts in response to M-CSF and RANKL. Administration of 5-fluorouracil to mice induces myelosuppression, but QuOPs survive and differentiate into osteoclasts in response to an active vitamin D3 analogue given to those mice. Mononuclear cells expressing c-Fms and RANK but not Ki67 are detected along bone surfaces in the vicinity of osteoblasts in RANKL-deficient mice. These results suggest that QuOPs preexist at the site of osteoclastogenesis and that osteoblasts are important for maintenance of QuOPs.

c-Src kinase is a rate-limiting activator of osteoclast (OC) function and Src inhibitors are therefore candidate antiosteoporosis drugs. By affecting [alpha]v[beta]3 and macrophage-colony stimulating factor (M-CSF)-induced signaling, c-Src is central to osteoclast activity, but not differentiation. We find Lyn, another member of Src family kinases (SFK) is, in contrast, a negative regulator of osteoclastic bone resorption. The absence of Lyn enhances receptor activator of NF-[kappa]B ligand (RANKL)-mediated differentiation of osteoclast precursors without affecting proliferation and survival, while its overexpression decreases osteoclast formation. In further contrast to c-Src, Lyn deficiency does not impact the activity of the mature cell. Reflecting increased osteoclast development in vitro, Lyn-/- mice undergo accelerated osteoclastogenesis and bone loss, in vivo, in response to RANKL. Mechanistically, Lyn forms a complex with receptor activator of NF-[kappa]B (RANK), the tyrosine phosphatase, SHP-1, and the adapter protein, Grb2-associated binder 2 (Gab2). Upon RANKL exposure, Gab2 phosphorylation, JNK, and NF-[kappa]B activation are enhanced in Lyn-/- osteoclasts, all critical events in osteoclast development. We therefore establish that Lyn regulates osteoclast formation and does it in a manner antithetical to that of c-Src. The most pragmatic aspect of our findings is that successful therapeutic inhibition of c-Src, in the context of the osteoclast, will require its stringent targeting. c-Src | osteoclast | SFK

Osteoclastogenesis is a tightly regulated biological process, and deregulation can lead to severe bone disorders such as osteoporosis. The regulation of osteoclastic signaling is incompletely understood, but ubiquitination of TNF receptor--associated factor 6 (TRAF6) has recently been shown to be important in mediating this process. We therefore investigated the role of the recently identified deubiquitinating enzyme CYLD in osteoclastogenesis and found that mice with a genetic deficiency of CYLD had aberrant osteoclast differentiation and developed severe osteoporosis. Cultured osteoclast precursors derived from CYLD-deficient mice were hyper-responsive to RANKL-induced differentiation and produced more and larger osteoclasts than did controls upon stimulation. We assessed the expression pattern of CYLD and found that it was drastically upregulated during RANKL-induced differentiation of preosteoclasts. Furthermore, CYLD negatively regulated RANK signaling by inhibiting TRAF6 ubiquitination and activation of downstream signaling events. Interestingly, we found that CYLD interacted physically with the signaling adaptor p62 and thereby was recruited to TRAF6. These findings establish CYLD as a crucial negative regulator of osteoclastogenesis and suggest its involvement in the p62/TRAF6 signaling axis., Introduction Normal bone physiology is regulated by the harmonic actions of osteoblasts and osteoclasts (OCs), cells that mediate bone formation and bone resorption, respectively (1). OCs are multinucleated cells that [...]