Relevance of an in vitro osteoclastogenesis system to study receptor activator of NF-kB ligand and osteoprotegerin biological activities

Receptor activator of NF-kB Ligand (RANKL) is an essential requirement for osteoclastogenesis and its activity is neutralized by binding to the soluble decoy receptor osteoprotegerin (OPG). The purpose of this work was to study the effects of RANKL and OPG during osteoclastogenesis using the murine monocytic cell line RAW 264.7 that can differentiate into osteoclasts in vitro. RAW 264.7 cells plated at 10(4) cells/cm(2) and cultured for 4 days in the presence of RANKL represent the optimal culture conditions for osteoclast differentiation, with an up-regulation of all parameters related to bone resorption: tartrate resistant acid phosphatase (TRAP), calcitonin receptor (CTR), RANK, cathepsin K, matrix metalloproteinase (MMP)-9 mRNA expressions. RANKL and OPG biological effects vary according to the differentiation state of the cells: in undifferentiated RAW 264.7 cells, TRAP expression was decreased by OPG and RANKL, RANK expression was inhibited by OPG, while MMP-9 and cathepsin K mRNA expressions were not modulated. In differentiated RAW 264.7 cells, RANKL and OPG both exert an overall inhibitory effect on the expression of all the parameters studied. In these experimental conditions, OPG-induced MMP-9 inhibition was abrogated in the presence of a blocking anti-RANKL antibody, suggesting that part of OPG effects are RANKL-dependent.     

Human growth hormone stimulates proteinase activities of rabbit bone cells via IGF-I

Human growth hormone (hGH) and human insulin-like growth factor-I (hIGF-I) are known to have a marked influence on osteoclastic formation and bone resorption in an unfractionated rabbit bone cell model. This study investigated the effects of both of these factors on the induction of cysteine-proteinases and matrix metalloproteinase-2 (MMP-2) and MMP-9. After 4 days of rabbit bone cell culture, hGH and hIGF-I significantly modulated cathepsin, MMP-9 (latent form) and MMP-2 (active form) activities. Similar studies were performed in the presence of parathyroid hormone (hPTH). hPTH increased MMP-2 and MMP-9 activities whereas it had no effect on the production of cathepsins by bone cells. When neutralizing anti-hIGF-1 antiserum was added to the culture, the stimulatory effects of hGH were totally abolished, indicating that hGH-modulated cathepsin and metalloproteinase activities were partly mediated by local hIGF-I secretion. Cysteine-proteinase activities released by purified osteoclasts were very low and were not modulated by hGH and h-IGF-I. However, hIGF-I but not hGH increased MMP-2 and MMP-9 activities released by purified osteoclasts. It may be concluded that hGH markedly stimulates the expression of proteinases in total rabbit bone cells via local hIGF-I production by stromal cells. Cysteine-proteinase activities are mainly produced by non-osteoclastic cells, while MMP-2 and MMP-9 modulated by hIGF-I are mainly expressed by osteoclastic cells.     

IL-17A suppresses the expression of bone resorption-related proteinases and osteoclast differentiation via IL-17RA or IL-17RC receptors in RAW264.7 cells

Interleukin-17 (IL-17) is produced exclusively by activated T cells and neutrophils, and stimulates osteoclastic bone resorption via osteoblasts by inducing the expression of “receptor activator of NF-κB (RANK) ligand” (RANKL). However, the direct effects of IL-17 on the differentiation of osteoclast precursors into osteoclasts and on the function of osteoclasts have not been clarified. Therefore, we examined the effects of IL-17A on the differentiation of osteoclast precursors using RAW264.7 cells and also on the expression of carbonic anhydrase II (CA II), cathepsin K, matrix metalloproteinases-9 (MMP-9), RANK, c-fms, and IL-17 receptors in these cells. The cells were cultured with or without 0.1, 1.0, 10 or 50 ng/mL IL-17 in the presence of soluble RANKL for up to 10 days. The CA II, cathepsin K, and MMP-9 mRNA and protein expression levels were examined using real-time PCR and Western blotting, respectively. The mRNA expression levels of RANK, c-fms, and IL-17 receptors were monitored by real-time PCR. Osteoclast differentiation was estimated using tartrate-resistant acid phosphatase (TRAP) staining of the cells. TRAP-positive cells were observed after day 5 of culture, and the number of cells decreased in the presence of 10 and 50 ng/mL IL-17A at days 5 and 7. In the presence of IL-17A, the expressions of cathepsin K, MMP-9 and c-fms decreased markedly on days 5 and/or 7 of culture, whereas the expression of CA II and IL-17 receptor (type A) increased remarkably at days 3 and 7, respectively. The expression of RANK and IL-17 receptor (type C) was not affected by the addition of IL-17A. These results suggest that the differentiation of osteoclast precursors into osteoclasts is suppressed at high concentrations of IL-17A. Furthermore, IL-17A suppresses the hydrolysis of matrix proteins during bone resorption by decreasing the production of cathepsin K and MMP-9 in osteoclasts.     

Cystatin B as an intracellular modulator of bone resorption

Degradation of organic bone matrix requires proteinase activity. Cathepsin K is a major osteoclast proteinase needed for bone resorption, although osteoclasts also express a variety of other cysteine- and matrix metalloproteinases that are involved in bone remodellation. Cystatin B, an intracellular cysteine proteinase inhibitor, exhibits a lysosomal distribution preferentially in osteoclasts but it's role in osteoclast physiology has remained unknown. The current paper describes a novel regulatory function for cystatin B in bone-resorbing osteoclasts in vitro. Rat osteoclasts were cultured on bovine bone and spleen-derived cystatin B was added to the cultures. Nuclear morphology was evaluated and the number of actively resorbing osteoclasts and resorption pits was counted. Intracellular cathepsin K and tartrate-resistant acid phosphatase (TRACP) activities were monitored using fluorescent enzyme substrates and immunohistology was used to evaluate distribution of cystatin B in rat metaphyseal bone. Microscopical evaluation showed that cystatin B inactivated osteoclasts, thus resulting in impaired bone resorption. Cathepsin K and TRACP positive vesicles disappeared dose-dependently from the cystatin B-treated osteoclasts, indicating a decreased intracellular trafficking of bone degradation products. At the same time, cystatin B protected osteoclasts from experimentally induced apoptosis. These data show for the first time that, in addition to regulating cysteine proteinase activity and promoting cell survival in the nervous system, cystatin B inhibits bone resorption by down-regulating intracellular cathepsin K activity despite increased osteoclast survival.     

A cytochemical assay for osteoclast cathepsin K activity

Cathepsin K is a member of the papain superfamily of cysteine proteases and plays a pivotal role in osteoclast-mediated bone resorption. This enzyme is an excellent target for antiresorptive therapies for osteopenic disorders such as osteoporosis.(1) Although isolated inhibitor studies on purified enzymes is required to discover potent and selective inhibitors of cathepsin K, a quantitative cytochemical assay(2) for cathepsin K would allow inhibitors to be tested on actual osteoclasts within sections of bone. Furthermore cathepsin K activity could be used to identify and analyse osteoclasts at definitive stages of their lifespan. A cytochemical assay is described that localizes osteoclast cathepsin K activity in unfixed, undecalcified cryostat sections of animal and human bone.     

Interleukin-17A induces cathepsin K and MMP-9 expression in osteoclasts via celecoxib-blocked prostaglandin E2 in osteoblasts

Interleukin-17 (IL-17) is a cytokine secreted primarily by T_H-17 cells that can stimulate the development of osteoclasts (osteoclastogenesis) in the presence of osteoblasts. IL-17, through osteoblasts, has indirect effects on the expression of bone resorption-related enzymes in osteoclasts, which have not been well clarified. Here, using MC3T3-E1 cells and RAW264.7 cells as osteoblasts and osteoclast precursors, we aimed to clarify these effects of IL-17A. MC3T3-E1 cells were cultured in the presence or absence of IL-17A for 72 h and the conditioned media collected (in the presence of soluble receptor activator of NF-кB ligand) and used to culture RAW264.7 cells. To assess osteoclast differentiation, adherent cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). Our analyses demonstrated that the number of TRAP-positive multinucleated cells increases after 3 days of culture in conditioned medium from IL-17A-treated cells compared to untreated controls. In addition, we observed that the levels of cathepsin K and MMP-9 increase in the conditioned medium from IL-17A-treated cells, whereas CA II expression levels remain unaffected. PGE₂ production from MC3T3-E1 cells increased in the presence of IL-17A. Celecoxib, a specific inhibitor of cyclooxygenase-2 (COX-2), blocked both the IL-17A-stimulated increase in TRAP-positive multinucleated cells and the expression of cathepsin K and MMP-9. Furthermore, when MC3T3-E1 cells were transformed with small interfering RNA to silence COX-2 expression before IL-17A treatment, the resulting conditioned medium was less effective at inducing cathepsin K and MMP-9 expression in RAW264.7 cells. These results suggest that IL-17A induces the differentiation and function of osteoclasts via celecoxib-blocked prostaglandin, mainly PGE₂, in osteoblasts.     

Regulation of osteoclast protease expression by RANKL

Receptor activator of NF-kappaB ligand (RANKL) is essential for osteoclast (OC) differentiation/activation and functions through its receptor RANK at the surface of the osteoclastic cells. This study investigated for the first time the direct effects of hRANKL on protease/protease inhibitor expressions and protease activities in purified rabbit osteoclast cultures, using semi-quantitative RT-PCR, gelatin zymography, and enzymatic assays. RANKL was shown to exert in vitro pro-resorptive effects by increasing osteoclast marker expressions (Tartrate resistant acid phosphatase (TRAP) and cathepsin K), MMP-9 expression, and pro-MMP-9 activity and by diminishing TIMP-1 expression, leading to an up-regulation of the MMP-9/TIMP-1 ratio.     

Fibroblast growth factor (FGF)-2 directly stimulates mature osteoclast function through activation of FGF receptor 1 and p42/p44 MAP kinase

We previously reported that fibroblast growth factor-2 (FGF-2) acts not only on osteoblasts to stimulate osteoclastic bone resorption indirectly but also on mature osteoclasts directly. In this study, we investigated the mechanism of this direct action of FGF-2 on mature osteoclasts using mouse and rabbit osteoclast culture systems. FGF-2 stimulated pit formation resorbed by isolated rabbit osteoclasts moderately from low concentrations (>/=10(-12) m), whereas at high concentrations (>/=10(-9) m) it showed stimulation on pit formation resorbed by unfractionated bone cells very potently. FGF-2 (>/=10(-12) m) also increased cathepsin K and MMP-9 mRNA levels in mouse and rabbit osteoclasts. Among FGF receptors (FGFR1 to 4) only FGFR1 was detected on isolated mouse osteoclasts, whereas all FGFRs were identified on mouse osteoblasts. FGF-2 (>/=10(-12) m) up-regulated the phosphorylation of cellular proteins, including p42/p44 mitogen-activated protein (MAP) kinase, and increased the kinase activity of immunoprecipitated FGFR1 in mouse osteoclasts. The stimulation of FGF-2 on mouse and rabbit osteoclast functions was abrogated by PD-98059, a specific inhibitor of p42/p44 MAP kinase. These results strongly suggest that FGF-2 acts directly on mature osteoclasts through activation of FGFR1 and p42/p44 MAP kinase, causing the stimulation of bone resorption at physiological or pathological concentrations.     

Purified human chondroitin-4-sulfate reduced MMP/TIMP imbalance induced by iron plus ascorbate in human fibroblast cultures

Imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) is an important control point in tissue remodelling. Several findings have reported a marked MMP/TIMP imbalance in a variety of in vitro models in which oxidative stress was induced. Since previous studies showed that commercial hyaluronan and chondroitin-4-sulphate are able to limit lipid peroxidation during oxidative stress, we investigated the antioxidant capacity of purified human plasma chondroitin-4-sulfate in reducing MMP and TIMP imbalance in a model of ROS-induced oxidative injury in fibroblast cultures. Purified human plasma chondroitin-4-sulfate was added to the fibroblast cultures exposed to FeSO4 plus ascorbate. We assayed cell death, MMP and TIMP mRNA expression and protein activities, DNA damage, membrane lipid peroxidation, and aconitase depletion. FeSO4 plus ascorbate produced severe death of cells and increased MMP-1, MMP-2 and MMP-9 expression and protein activities. It also caused DNA strand breaks, enhanced lipid peroxidation and decreased aconitase. TIMP-1 and TIMP-2 protein levels and mRNA expression remain unaltered. Purified human plasma C4S, at three different doses, restored the MMP/TIMP homeostasis, increased cell survival, reduced DNA damage, inhibited lipid peroxidation and limited impairment of aconitase. These results further support the hypothesis that these biomolecules possess antioxidant activity and by reducing ROS production C4S may limit cell injury produced by MMP/TIMP imbalance.     

Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones

Bone development requires the recruitment of osteoclast precursors from surrounding mesenchyme, thereby allowing the key events of bone growth such as marrow cavity formation, capillary invasion, and matrix remodeling. We demonstrate that mice deficient in gelatinase B/matrix metalloproteinase (MMP)-9 exhibit a delay in osteoclast recruitment. Histological analysis and specialized invasion and bone resorption models show that MMP-9 is specifically required for the invasion of osteoclasts and endothelial cells into the discontinuously mineralized hypertrophic cartilage that fills the core of the diaphysis. However, MMPs other than MMP-9 are required for the passage of the cells through unmineralized type I collagen of the nascent bone collar, and play a role in resorption of mineralized matrix. MMP-9 stimulates the solubilization of unmineralized cartilage by MMP-13, a collagenase highly expressed in hypertrophic cartilage before osteoclast invasion. Hypertrophic cartilage also expresses vascular endothelial growth factor (VEGF), which binds to extracellular matrix and is made bioavailable by MMP-9 (Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Nat. Cell Biol. 2:737-744). We show that VEGF is a chemoattractant for osteoclasts. Moreover, invasion of osteoclasts into the hypertrophic cartilage requires VEGF because it is inhibited by blocking VEGF function. These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.     

Nicotine Affects Bone Resorption and Suppresses the Expression of Cathepsin K,MMP-9 and Vacuolar-Type H+-ATPase d2 and Actin Organization in Osteoclasts

Tobacco smoking is an important risk factor for the development of several cancers, osteoporosis, and inflammatory diseases such as periodontitis. Nicotine is one of the major components of tobacco. In previous study, we showed that nicotine inhibits mineralized nodule formation by osteoblasts, and the culture medium from osteoblasts containing nicotine and lipopolysaccharide increases osteoclast differentiation. However, the direct effect of nicotine on the differentiation and function of osteoclasts is poorly understood. Thus, we examined the direct effects of nicotine on the expression of nicotine receptors and bone resorption-related enzymes, mineral resorption, actin organization, and bone resorption using RAW264.7 cells and bone marrow cells as osteoclast precursors. Cells were cultured with 10⁻⁵, 10⁻⁴, or 10⁻³ M nicotine and/or 50 µM α-bungarotoxin (btx), an 7 nicotine receptor antagonist, in differentiation medium containing the soluble RANKL for up 7 days. 1–5, 7, 9, and 10 nicotine receptors were expressed on RAW264.7 cells. The expression of 7 nicotine receptor was increased by the addition of nicotine. Nicotine suppressed the number of tartrate-resistant acid phosphatase positive multinuclear osteoclasts with large nuclei(≥10 nuclei), and decreased the planar area of each cell. Nicotine decreased expression of cathepsin K, MMP-9, and V-ATPase d2. Btx inhibited nicotine effects. Nicotine increased CA II expression although decreased the expression of V-ATPase d2 and the distribution of F-actin. Nicotine suppressed the planar area of resorption pit by osteoclasts, but did not affect mineral resorption. These results suggest that nicotine increased the number of osteoclasts with small nuclei, but suppressed the number of osteoclasts with large nuclei. Moreover, nicotine reduced the planar area of resorption pit by suppressing the number of osteoclasts with large nuclei, V-ATPase d2, cathepsin K and MMP-9 expression and actin organization.     

Induction of osteoclast differentiation by Runx2 through receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin regulation and partial rescue of osteoclastogenesis in Runx2-/- mice by RANKL transgene

Receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), and macrophage-colony stimulating factor play essential roles in the regulation of osteoclastogenesis. Runx2-deficient (Runx2-/-) mice showed a complete lack of bone formation because of maturational arrest of osteoblasts and disturbed chondrocyte maturation. Further, osteoclasts were absent in these mice, in which OPG and macrophage-colony stimulating factor were normally expressed, but RANKL expression was severely diminished. We investigated the function of Runx2 in osteoclast differentiation. A Runx2-/- calvaria-derived cell line (CA120-4), which expressed OPG strongly but RANKL barely, severely suppressed osteoclast differentiation from normal bone marrow cells in co-cultures. Adenoviral introduction of Runx2 into CA120-4 cells induced RANKL expression, suppressed OPG expression, and restored osteoclast differentiation from normal bone marrow cells, whereas the addition of OPG abolished the osteoclast differentiation induced by Runx2. Addition of soluble RANKL (sRANKL) also restored osteoclast differentiation in co-cultures. Forced expression of sRANKL in Runx2-/- livers increased the number and size of osteoclast-like cells around calcified cartilage, although vascular invasion into the cartilage was superficial because of incomplete osteoclast differentiation. These findings indicate that Runx2 promotes osteoclast differentiation by inducing RANKL and inhibiting OPG. As the introduction of sRANKL was insufficient for osteoclast differentiation in Runx2-/- mice, however, our findings also suggest that additional factor(s) or matrix protein(s), which are induced in terminally differentiated chondrocytes or osteoblasts by Runx2, are required for osteoclastogenesis in early skeletal development.     

Blockade of the sympathetic nervous system degrades ligament in a rat MCL model.

We hypothesize that blockade of the sympathetic nervous system degrades ligament. We tested this hypothesis in a rat medial collateral ligament (MCL) model. Fifteen animals were treated for 10 days with the sympathetic chemotoxin guanethidine using osmotic pumps, whereas 15 control rats received pumps containing saline. A reduction in plasma concentrations of norepinephrine in the guanethidine rats indicated a significant decrease in sympathetic nerve activity. Vasoactive intestinal peptide and neuropeptide Y were decreased in MCLs from guanethidine animals, as quantified by radioimmunoassays. Tissue vascularity was substantially increased in guanethidine MCLs, whereas mechanical properties were significantly decreased. Proteases, such as matrix metalloproteinases (MMP) and cysteine proteases, play a major role in ligament degradation. The proteases MMP-13, cathepsin K, and tartrate-resistant acid phosphatase (TRAP) have collagenolytic activity and have been shown in rat ligament tissues. To determine whether the degradation seen in this study was due to protease activity, we determined the expression of these enzymes in control and treated MCLs. Real-time quantitative PCR revealed that guanethidine treatment increased expression of MMP-13 and cathepsin K mRNAs, although overall expression levels of MMP-13 and TRAP were relatively low. Histology also identified increases in TRAP and cathepsin K, but not MMP-13, in guanethidine-treated tissues. Results support our hypothesis that blockade of the sympathetic nervous system substantially degrades ligament.     

Functional heterogeneity of osteoclasts: matrix metalloproteinases participate in osteoclastic resorption of calvarial bone but not in resorption of long bone.

Data in the literature suggest that site-specific differences exist in the skeleton with respect to digestion of bone by osteoclasts. Therefore, we investigated whether bone resorption by calvarial osteoclasts (intramembranous bone) differs from resorption by long bone osteoclasts (endochondral bone). The involvement of two major classes of proteolytic enzymes, the cysteine proteinases (CPs) and matrix metalloproteinases (MMPs), was studied by analyzing the effects of selective low molecular weight inhibitors of these enzymes on bone resorption. Mouse tissue explants (calvariae and long bones) as well as rabbit osteoclasts, which had been isolated from both skeletal sites and subsequently seeded on bone slices, were cultured in the presence of inhibitors and resorption was analyzed. The activity of the CP cathepsins B and K and of MMPs was determined biochemically (CPs and MMPs) and enzyme histochemically (CPs) in explants and isolated osteoclasts. We show that osteoclastic resorption of calvarial bone depends on activity of both CPs and MMPs, whereas long bone resorption depends on CPs, but not on the activity of MMPs. Furthermore, significantly higher levels of cathepsin B and cathepsin K activities were expressed by long bone osteoclasts than by calvarial osteoclasts. Resorption of slices of bovine skull or cortical bone by osteoclasts isolated from long bones was not affected by MMP inhibitors, whereas resorption by calvarial osteoclasts was inhibited. Inhibition of CP activity affected the resorption by the two populations of osteoclasts in a similar way. We conclude that this is the first report to show that significant differences exist between osteoclasts of calvariae and long bones with respect to their bone resorbing activities. Resorption by calvarial osteoclasts depends on the activity of CPs and MMPs, whereas resorption by long bone osteoclasts depends primarily on the activity of CPs. We hypothesize that functionally different subpopulations of osteoclasts, such as those described here, originate from different sets of progenitors.     

Cathepsin K Activity-dependent Regulation of Osteoclast Actin Ring Formation and Bone Resorption

Cathepsin K is responsible for the degradation of type I collagen in osteoclast-mediated bone resorption. Collagen fragments are known to be biologically active in a number of cell types. Here, we investigate their potential to regulate osteoclast activity. Mature murine osteoclasts were seeded on type I collagen for actin ring assays or dentine discs for resorption assays. Cells were treated with cathepsins K-, L-, or MMP-1-predigested type I collagen or soluble bone fragments for 24 h. The presence of actin rings was determined fluorescently by staining for actin. We found that the percentage of osteoclasts displaying actin rings and the area of resorbed dentine decreased significantly on addition of cathepsin K-digested type I collagen or bone fragments, but not with cathepsin L or MMP-1 digests. Counterintuitively, actin ring formation was found to decrease in the presence of the cysteine proteinase inhibitor LHVS and in cathepsin K-deficient osteoclasts. However, cathepsin L deficiency or the general MMP inhibitor GM6001 had no effect on the presence of actin rings. Predigestion of the collagen matrix with cathepsin K, but not by cathepsin L or MMP-1 resulted in an increased actin ring presence in cathepsin K-deficient osteoclasts. These studies suggest that cathepsin K interaction with type I collagen is required for 1) the release of cryptic Arg-Gly-Asp motifs during the initial attachment of osteoclasts and 2) termination of resorption via the creation of autocrine signals originating from type I collagen degradation.Osteoclasts are monocyte-macrophage lineage-derived, large multinucleated cells. They are the major bone resorbing cells, essential for bone turnover and development. Active osteoclasts display characteristic membranes, including the ruffled border, attachment zone, and the basolateral secretory membrane. After attachment to bone, the ruffled border secretes enzymes and protons enabling the solubilization and digestion of the bone matrix. Osteoclasts express many proteases including cathepsins and matrix metalloproteases (MMPs)² (for review see Refs. 1-3). However, it is the general consensus that cathepsin K (catK) is the major bone-degrading enzyme (4-7).Rapid cytoskeletal reorganization is essential for osteoclast function and formation of the specialized membranes. Bone resorption occurs within the sealing zone, which is formed by an actin ring structure. This can be identified as a solid circular belt like formation and consists of an actin filament core surrounded by actin-binding proteins such as talin, α-actinin, and vinculin, which link matrix-recognizing integrins to the cytoskeleton (8). The ruffled border is contained within this structure. The actin ring is initiated by the formation of podosomes, which represent dot-like actin structures of small F-actin containing columns surrounded by proteins also found in focal adhesion such as vinculin and paxillin (9). It was previously thought that the sealing zone was formed by the fusion of podosomes after the osteoclast becomes activated (10, 11), but it has since been demonstrated that podosomes and the sealing zone are distinct structures (12, 13). It should be noted that bone resorption only occurs when the sealing zone is formed and the actin ring is present (14).Osteoclasts bind and interact with the bone surface through specific integrin receptors. The most abundant integrin present in osteoclasts is the αvß3 receptor also known as the vitronectin receptor (15, 16). This receptor attaches to RGD sequence containing components of the bone matrix, e.g. vitronectin, osteopontin, and type I collagen (17-19). This interaction enables the formation and regulation of the actin ring and therefore osteoclast activity (20-22). It has previously been shown that soluble RGD containing peptides added to cell supernatant are capable of inhibiting osteoclast binding and bone resorption (18, 22-24).This study investigates the effect of collagen degradation fragments on osteoclast activity. Soluble type I collagen and the bone powder of murine long bones were subjected to digestion reactions by the cysteine proteases, catK and catL, and the interstitial collagenase, MMP-1. The effect of these degradation products on osteoclasts was investigated by monitoring actin ring and resorption pit formation. We further investigated the role of cathepsins using catK- and catL-deficient mice. Finally, we looked in more detail at the effect of collagen, as a cell adhesion matrix, on osteoclast activity.     

Dental pulp–derived stem cells inhibit osteoclast differentiation by secreting osteoprotegerin and deactivating AKT signalling in myeloid cells

Osteoclasts (OCs) differentiate from the monocyte/macrophage lineage, critically regulate bone resorption and remodelling in both homeostasis and pathology. Various immune and non-immune cells help initiating activation of myeloid cells for differentiation, whereas hyper-activation leads to pathogenesis, and mechanisms are yet to be completely understood. Herein, we show the efficacy of dental pulp–derived stem cells (DPSCs) in limiting RAW 264.7 cell differentiation and underlying molecular mechanism, which has the potential for future therapeutic application in bone-related disorders. We found that DPSCs inhibit induced OC differentiation of RAW 264.7 cells when co-cultured in a contact-free system. DPSCs reduced expression of key OC markers, such as NFATc1, cathepsin K, TRAP, RANK and MMP-9 assessed by quantitative RT-PCR, Western blotting and immunofluorescence detection methods. Furthermore, quantitative RT-PCR analysis revealed that DPSCs mediated M2 polarization of RAW 264.7 cells. To define molecular mechanisms, we found that osteoprotegerin (OPG), an OC inhibitory factor, was up-regulated in RAW 264.7 cells in the presence of DPSCs. Moreover, DPSCs also constitutively secrete OPG that contributed in limiting OC differentiation. Finally, the addition of recombinant OPG inhibited OC differentiation in a dose-dependent manner by reducing the expression of OC differentiation markers, NFATc1, cathepsin K, TRAP, RANK and MMP9 in RAW 264.7 cells. RNAKL and M-CSF phosphorylate AKT and activate PI3K-AKT signalling pathway during osteoclast differentiation. We further confirmed that OPG-mediated inhibition of the downstream activation of PI3K-AKT signalling pathway was similar to the DPSC co-culture–mediated inhibition of OC differentiation. This study provides novel evidence of DPSC-mediated inhibition of osteoclastogenesis mechanisms.     

Augmented LPS responsiveness in type 1 diabetes‐derived osteoclasts

Bone abnormalities are frequent co‐morbidities of type 1 diabetes (T1D) and are principally mediated by osteoblasts and osteoclasts which in turn are regulated by immunologic mediators. While decreased skeletal health in T1D involves alterations in osteoblast maturation and function, the effect of altered immune function on osteoclasts in T1D‐associated bone and joint pathologies is less understood. Here T1D‐associated osteoclast‐specific differentiation and function in the presence and absence of inflammatory mediators was characterized utilizing bone marrow‐derived osteoclasts (BM‐OCs) isolated from non‐obese diabetic (NOD) mice, a model for spontaneous autoimmune diabetes with pathology similar to individuals with T1D. Differentiation and osteoclast‐mediated bone resorption were evaluated along with cathepsin K, MMP‐9, and immune soluble mediator expression. The effect of lipopolysaccharide (LPS), a pro‐inflammatory cytokine cocktail, and NOD‐derived conditioned supernatants on BM‐OC function was also determined. Although NOD BM‐OCs cultures contained smaller osteoclasts, they resorbed more bone concomitant with increased cathepsin K, MMP‐9, and pro‐osteoclastogenic mediator expression. NOD BM‐OCs also displayed an inhibition of LPS‐induced deactivation that was not a result of soluble mediators produced by NOD BM‐OCs, although a pro‐inflammatory milieu did enhance NOD BM‐OCs bone resorption. Together these data indicate that osteoclasts from a T1D mouse model hyper‐respond to RANK‐L resulting in excessive bone degradation via enhanced cathepsin K and MMP‐9 secretion concomitant with an increased expression of pro‐osteoclastic soluble mediators. Our data also suggest that inhibition of LPS‐induced deactivation in NOD‐derived BM‐OC cultures is most likely due to NOD osteoclast responsiveness rather than LPS‐induced expression of soluble mediators. J. Cell. Physiol. 228: 349–361, 2013. © 2012 Wiley Periodicals, Inc.