PKA-induced Receptor Activator of NF-κB Ligand (RANKL) Expression in Vascular Cells Mediates Osteoclastogenesis but Not Matrix Calcification

Vascular calcification is a predictor of cardiovascular mortality and is prevalent in patients with atherosclerosis and chronic renal disease. It resembles skeletal osteogenesis, and many bone cells as well as bone-related factors involved in both formation and resorption have been localized in calcified arteries. Previously, we showed that aortic medial cells undergo osteoblastic differentiation and matrix calcification both spontaneously and in response to PKA agonists. The PKA signaling pathway is also involved in regulating bone resorption in skeletal tissue by stimulating osteoblast-production of osteoclast regulating cytokines, including receptor-activator of nuclear κB ligand (RANKL) and interleukins. Therefore, we investigated whether PKA activators regulate osteoclastogenesis in aortic smooth muscle cells (SMC). Treatment of murine SMC with the PKA agonist forskolin stimulated RANKL expression at both mRNA and protein levels. Forskolin also stimulated expression of interleukin-6 but not osteoprotegerin (OPG), an inhibitor of RANKL. Consistent with these results, osteoclastic differentiation was induced when monocytic preosteoclasts (RAW264.7) were cocultured with forskolin-treated aortic SMC. Oxidized phospholipids also slightly induced RANKL expression in T lymphocytes, another potential source of RANKL in the vasculature. Because previous studies have shown that RANKL treatment alone induces matrix calcification of valvular and vascular cells, we next examined whether RANKL mediates forskolin-induced matrix calcification by aortic SMC. RANKL inhibition with OPG had little or no effect on osteoblastic differentiation and matrix calcification of aortic SMC. These findings suggest that, as in skeletal tissues, PKA activation induces bone resorptive factors in the vasculature and that aortic SMC calcification specifically induced by PKA, is not mediated by RANKL.     

Porphyromonas gingivalis Stimulates Bone Resorption by Enhancing RANKL (Receptor Activator of NF-κB Ligand) through Activation of Toll-like Receptor 2 in Osteoblasts

Periodontitis has been associated with rheumatoid arthritis. In experimental arthritis, concomitant periodontitis caused by oral infection with Porphyromonas gingivalis enhances articular bone loss. The aim of this study was to investigate how lipopolysaccharide (LPS) from P. gingivalis stimulates bone resorption. The effects by LPS P. gingivalis and four other TLR2 ligands on bone resorption, osteoclast formation, and gene expression in wild type and Tlr2-deficient mice were assessed in ex vivo cultures of mouse parietal bones and in an in vivo model in which TLR2 agonists were injected subcutaneously over the skull bones. LPS P. gingivalis stimulated mineral release and matrix degradation in the parietal bone organ cultures by increasing differentiation and formation of mature osteoclasts, a response dependent on increased RANKL (receptor activator of NF-κB ligand). LPS P. gingivalis stimulated RANKL in parietal osteoblasts dependent on the presence of TLR2 and through a MyD88 and NF-κB-mediated mechanism. Similarly, the TLR2 agonists HKLM, FSL1, Pam2, and Pam3 stimulated RANKL in osteoblasts and parietal bone resorption. LPS P. gingivalis and Pam2 robustly enhanced osteoclast formation in periosteal/endosteal cell cultures by increasing RANKL. LPS P. gingivalis and Pam2 also up-regulated RANKL and osteoclastic genes in vivo, resulting in an increased number of periosteal osteoclasts and immense bone loss in wild type mice but not in Tlr2-deficient mice. These data demonstrate that LPS P. gingivalis stimulates periosteal osteoclast formation and bone resorption by stimulating RANKL in osteoblasts via TLR2. This effect might be important for periodontal bone loss and for the enhanced bone loss seen in rheumatoid arthritis patients with concomitant periodontal disease.     

An Inducible,Ligand-Independent Receptor Activator of NF-κB Gene to Control Osteoclast Differentiation from Monocytic Precursors

Osteoclasts are bone-resorbing cells that are critical for the normal formation and maintenance of teeth and skeleton. Osteoclast deficiency can contribute to heterotopic ossification (HO), a pathology that is particularly detrimental to the mechanical functions of joints, valves and blood vessels. On the other hand, osteoclast over-activity is a major cause of osteoporosis. A reliable method for controlled generation of osteoclasts would be useful as a potential autologous cell therapy for HO, as well as high-throughput drug screening for anti-osteoporotic drugs. In this report, we describe the development of a cell engineering approach to control monocytic precursor cell differentiation to osteoclasts. Oligomerization of receptor activator of nuclear factor κB (RANK) is known to be essential for osteoclast differentiation from monocyte/macrophage precursors. We engineered a murine monocytic cell line, RAW264.7 to express a fusion protein comprising the intracellular RANK signaling domain and FK506-derived dimerization domains that bind to a small molecule chemical inducer of dimerization (CID). Virally infected cells expressing this fusion protein were treated with CID and dose-dependent induction of tartrate-resistant acid phosphatase activity, as well as multinucleated osteoclast formation were observed. Furthermore, NF-κB signaling was upregulated in a CID-dependent fashion, demonstrating effective RANK intracellular signaling. Functionally CID-induced osteoclasts had robust mineral resorptive activity in both two-dimensional and three-dimensional in vitro resorption assays. In addition, the CID-induced osteoclasts have the same life span as native RANKL-induced osteoclasts. Most importantly and crucially, the engineered cells differentiated into osteoclasts that were resistant to the potent osteoclast inhibitor, osteoprotegerin. Taken together, these studies are the first to describe a method for inducible control of monocytic precursor differentiation to osteoclasts that may be useful for future development of an engineered autologous cell therapy as well as high-throughput drug testing systems to treat diseases of osteoclast over-activity that are independent of osteoprotegerin.     

Molecular cloning and functional characterization of the porcine extracellular domain of Receptor Activator of NF-κB Ligand (sRANKL)

Receptor Activator of NF-κB Ligand (RANKL) plays a pivotal role as a regulator of osteoclast activity and is involved in osteoporosis. Here, we report the cloning and functional characterization of the complete extracellular domain of the porcine RANKL gene (sRANKL). The porcine sRANKL cDNA has an ORF of 744 nucleotides and shares 87%, 80% and 80% identity with human, rat and mouse RANKL coding sequences, respectively. The protein consists of 247 amino acids with 90%, 81% and 80% sequences similarities compared to human, mouse and rat RANKL, respectively. Over-expression of porcine sRANKL led to osteoclast formation. The osteoclasts showed a characteristic morphology, expressed the carbonic anhydrase type 2, were TRACP positive and exhibited a bone-resorbing activity.In conclusion, we first describe the molecular cloning and functional characterization of porcine sRANKL, which will help to understand the function of a RANKL gene in large animal models.     

TRAF Family Member-Associated NF-��B Activator (TANK) Induced by RANKL Negatively Regulates Osteoclasts Survival and Function

Osteoclasts are the principle bone-resorbing cells. Precise control of balanced osteoclast activity is indispensable for bone homeostasis. Osteoclast activation mediated by RANK-TRAF6 axis has been clearly identified. However, a negative regulation-machinery in osteoclast remains unclear. TRAF family member-associated NF-κB activator (TANK) is induced by about 10 folds during osteoclastogenesis, according to a genome-wide analysis of gene expression before and after osteoclast maturation, and confirmed by western blot and quantitative RT-PCR. Bone marrow macrophages (BMMs) transduced with lentivirus carrying tank-shRNA were induced to form osteoclast in the presence of RANKL and M-CSF. Tank expression was downregulated by 90% by Tank-shRNA, which is confirmed by western blot. Compared with wild-type (WT) cells, osteoclastogenesis of Tank-silenced BMMs was increased, according to tartrate-resistant acid phosphatase (TRAP) stain on day 5 and day 7. Number of bone resorption pits by Tank-silenced osteoclasts was increased by 176% compared with WT cells, as shown by wheat germ agglutinin (WGA) stain and scanning electronic microscope (SEM) analysis. Survival rate of Tank-silenced mature osteoclast is also increased. However, acid production of Tank-knockdown cells was not changed compared with control cells. IκBα phosphorylation is increased in tank-silenced cells, indicating that TANK may negatively regulate NF-κB activity in osteoclast. In conclusion, Tank, whose expression is increased during osteoclastogenesis, inhibits osteoclast formation, activity and survival, by regulating NF-κB activity and c-FLIP expression. Tank enrolls itself in a negative feedback loop in bone resorption. These results may provide means for therapeutic intervention in diseases of excessive bone resorption.     

Receptor Activator of Nuclear Factor κB Ligand (RANKL) Protein Expression by B Lymphocytes Contributes to Ovariectomy-induced Bone Loss

Production of the cytokine receptor activator of NFκB ligand (RANKL) by lymphocytes has been proposed as a mechanism by which sex steroid deficiency causes bone loss. However, there have been no studies that functionally link RANKL expression in lymphocytes with bone loss in this condition. Herein, we examined whether RANKL expression in either B or T lymphocytes contributes to ovariectomy-induced bone loss in mice. Mice harboring a conditional RANKL allele were crossed with CD19-Cre or Lck-Cre mice to delete RANKL in B or T lymphocytes, respectively. Deletion of RANKL from either cell type had no impact on bone mass in estrogen-replete mice up to 7 months of age. However, mice lacking RANKL in B lymphocytes were partially protected from the bone loss caused by ovariectomy. This protection occurred in cancellous, but not cortical, bone and was associated with a failure to increase osteoclast numbers in the conditional knock-out mice. Deletion of RANKL from T lymphocytes had no impact on ovariectomy-induced bone loss. These results demonstrate that lymphocyte RANKL is not involved in basal bone remodeling, but B cell RANKL does contribute to the increase in osteoclasts and cancellous bone loss that occurs after loss of estrogen.     

Stimulation of Bone Formation in Cortical Bone of Mice Treated with a Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-binding Peptide That Possesses Osteoclastogenesis Inhibitory Activity

To date, parathyroid hormone is the only clinically available bone anabolic drug. The major difficulty in the development of such drugs is the lack of clarification of the mechanisms regulating osteoblast differentiation and bone formation. Here, we report a peptide (W9) known to abrogate osteoclast differentiation in vivo via blocking receptor activator of nuclear factor-κB ligand (RANKL)-RANK signaling that we surprisingly found exhibits a bone anabolic effect in vivo. Subcutaneous administration of W9 three times/day for 5 days significantly augmented bone mineral density in mouse cortical bone. Histomorphometric analysis showed a decrease in osteoclastogenesis in the distal femoral metaphysis and a significant increase in bone formation in the femoral diaphysis. Our findings suggest that W9 exerts bone anabolic activity. To clarify the mechanisms involved in this activity, we investigated the effects of W9 on osteoblast differentiation/mineralization in MC3T3-E1 (E1) cells. W9 markedly increased alkaline phosphatase (a marker enzyme of osteoblasts) activity and mineralization as shown by alizarin red staining. Gene expression of several osteogenesis-related factors was increased in W9-treated E1 cells. Addition of W9 activated p38 MAPK and Smad1/5/8 in E1 cells, and W9 showed osteogenesis stimulatory activity synergistically with BMP-2 in vitro and ectopic bone formation. Knockdown of RANKL expression in E1 cells reduced the effect of W9. Furthermore, W9 showed a weak effect on RANKL-deficient osteoblasts in alkaline phosphatase assay. Taken together, our findings suggest that this peptide may be useful for the treatment of bone diseases, and W9 achieves its bone anabolic activity through RANKL on osteoblasts accompanied by production of several autocrine factors.     

Inhibition of Osteocyte Apoptosis Prevents the Increase in Osteocytic Receptor Activator of Nuclear Factor κB Ligand (RANKL) but Does Not Stop Bone Resorption or the Loss of Bone Induced by Unloading

Apoptosis of osteocytes and osteoblasts precedes bone resorption and bone loss with reduced mechanical stimulation, and receptor activator of NF-κB ligand (RANKL) expression is increased with unloading in mice. Because osteocytes are major RANKL producers, we hypothesized that apoptotic osteocytes signal to neighboring osteocytes to increase RANKL expression, which, in turn, increases osteoclastogenesis and bone resorption. The traditional bisphosphonate (BP) alendronate (Aln) or IG9402, a BP analog that does not inhibit resorption, prevented the increase in osteocyte apoptosis and osteocytic RANKL expression. The BPs also inhibited osteoblast apoptosis but did not prevent the increase in osteoblastic RANKL. Unloaded mice exhibited high serum levels of the bone resorption marker C-telopeptide fragments of type I collagen (CTX), elevated osteoclastogenesis, and increased osteoclasts in bone. Aln, but not IG9402, prevented all of these effects. In addition, Aln prevented the reduction in spinal and femoral bone mineral density, spinal bone volume/tissue volume, trabecular thickness, mechanical strength, and material strength induced by unloading. Although IG9402 did not prevent the loss of bone mass, it partially prevented the loss of strength, suggesting a contribution of osteocyte viability to strength independent of bone mass. These results demonstrate that osteocyte apoptosis leads to increased osteocytic RANKL. However, blockade of these events is not sufficient to restrain osteoclast formation, inhibit resorption, or stop bone loss induced by skeletal unloading.     

Receptor Activator of NF-κB (RANK) Cytoplasmic IVVY535–538 Motif Plays an Essential Role in Tumor Necrosis Factor-α (TNF)-mediated Osteoclastogenesis

Tumor necrosis factor-α (TNF) enhances osteoclast formation and activity leading to bone loss in various pathological conditions, but its precise role in osteoclastogenesis remains controversial. Although several groups showed that TNF can promote osteoclastogenesis independently of the receptor activator of NF-κB (RANK) ligand (RANKL), others demonstrated that TNF-mediated osteoclastogenesis needs permissive levels of RANKL. Here, we independently reveal that although TNF cannot stimulate osteoclastogenesis on bone slices, it can induce the formation of functional osteoclasts on bone slices in the presence of permissive levels of RANKL or from bone marrow macrophages (BMMs) pretreated by RANKL. TNF can still promote the formation of functional osteoclasts 2 days after transient RANKL pretreatment. These data have confirmed that TNF-mediated osteoclastogenesis requires priming of BMMs by RANKL. Moreover, we investigated the molecular mechanism underlying the dependence of TNF-mediated osteoclastogenesis on RANKL. RANK, the receptor for RANKL, contains an IVVY^535–538 motif that has been shown to play a vital role in osteoclastogenesis by committing BMMs to the osteoclast lineage. We show that TNF-induced osteoclastogenesis depends on RANKL to commit BMMs to the osteoclast lineage and RANKL regulates the lineage commitment through the IVVY motif. Mechanistically, the IVVY motif controls the lineage commitment by reprogramming osteoclast genes into an inducible state in which they can be activated by TNF. Our findings not only provide important mechanistic insights into the action of RANKL in TNF-mediated osteoclastogenesis but also establish that the IVVY motif may serve as an attractive therapeutic target for bone loss in various bone disorders.     

TRAF6 Autoubiquitination-Independent Activation of the NFκB and MAPK Pathways in Response to IL-1 and RANKL

The adapter protein TRAF6 is critical for mediating signal transduction from members of the IL-1R/TLR and TNFR superfamilies. The TRAF6 RING finger domain functions as an ubiquitin E3 ligase capable of generating non-degradative K63-linked ubiquitin chains. It is believed that these chains serve as docking sites for formation of signaling complexes, and that K63-linked autoubiquitination of TRAF6 is essential for formation and activation of a complex involving the kinase TAK1 and its adapters, TAB1 and TAB2. In order to assess independently the E3 ligase and ubiquitin substrate functions of TRAF6, we generated, respectively, RING domain and complete lysine-deficient TRAF6 mutants. We found that while the TRAF6 RING domain is required for activation of TAK1, it is dispensable for interaction between TRAF6 and the TAK1-TAB1-TAB2 complex. Likewise, lysine-deficient TRAF6 was found to interact with the TAK1-TAB1-TAB2 complex, but surprisingly was also found to be fully competent to activate TAK1, as well as NFκB and AP-1 reporters. Furthermore, lysine-deficient TRAF6 rescued IL-1-mediated NFκB and MAPK activation, as well as IL-6 elaboration in retrovirally-rescued TRAF6-deficient fibroblasts. Lysine-deficient TRAF6 also rescued RANKL-mediated NFκB and MAPK activation, and osteoclastogenesis in retrovirally-rescued TRAF6-deficient bone marrow macrophages. While incapable of being ubiquitinated itself, we demonstrate that lysine-deficient TRAF6 remains competent to induce ubiquitination of IKKγ/NEMO. Further, this NEMO modification contributes to TRAF6-mediated activation of NFκB. Collectively, our results suggest that while TRAF6 autoubiquitination may serve as a marker of activation, it is unlikely to underpin RING finger-dependent TRAF6 function.     

p53-mediated induction of Noxa and p53AIP1 requires NFκB

The intricate regulation of cell survival and cell death is critical for the existence of both normal and transformed cells. Two factors central to these processes are p53 and NFκB, with both factors having ascribed roles in both promoting and repressing cell death. Not surprisingly, a number of studies have previously reported interplay between p53 and NFκB. The mechanistic basis behind these observations, however, is currently incomplete. We report here further insights into this interplay using a system where blockade of NFκB inhibits cell death from p53, but at the same time sensitizes cells to death by TNFα. We found in agreement with a recent report showing that NFκB is required for the efficient activation of the BH3-only protein Noxa by the p53 family member p73, that p53’s ability to induce Noxa is also impeded by inhibition of NFκB. In contrast to the regulation by p73, however, blockade of NFκB downstream of p53 decreases Noxa protein levels without effects on Noxa mRNA. Our further analysis of the effects of NFκB inhibition on p53 target gene expression revealed that while most target genes analysed where unaffected by blockade of NFκB, the p53-mediated induction of the pro-apoptotic gene p53AIP1 was significantly dependent on NFκB. These studies therefore add further insight into the complex relationship of p53 and NFκB and since both Noxa and p53AIP1 have been shown to be important components of p53-mediated cell death responses, these findings may also indicate critical points where NFκB plays a pro-apoptotic role downstream of p53.     

TRPV-5 Mediates a Receptor Activator of NF-κB (RANK) Ligand-induced Increase in Cytosolic Ca2+ in Human Osteoclasts and Down-regulates Bone Resorption

Most of the signaling effectors located downstream of receptor activator of NF-κB (RANK) activation are calcium-sensitive. However, the early signaling events that lead to the mobilization of intracellular calcium in human osteoclasts are still poorly understood. The Ca²⁺-sensitive fluorescent probe Fura2 was used to detect changes in the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) in a model of human osteoclasts. Stimulating these cells with receptor activator of NF-κB ligand (RANKL) induced a rapid and significant increase in [Ca²⁺]_i. Adding extracellular Ca²⁺ chelators, depleting intracellular stores, and the use of a phospholipase C inhibitor all indicated that the Ca²⁺ was of extracellular origin, suggesting the involvement of a Ca²⁺ channel. We showed that none of the classical Ca²⁺ channels (L-, T-, or R-type) were involved in the RANKL-induced Ca²⁺ spike. However, the effect of high doses of Gd³⁺ did suggest that TRP family channels were present in human osteoclasts. The TRPV-5 channel was expressed in osteoclasts and was mainly located in the cellular area in contact with the bone surface. Furthermore, the RNA inactivation of TRPV-5 channel completely inhibited the RANKL-induced increase in [Ca²⁺]_i, which was accompanied in the long term by marked activation of bone resorption. Overall, our results show that RANKL induced a significant increase in [Ca²⁺]_i of extracellular origin, probably as a result of the opening of TRPV-5 calcium channels on the surface of human osteoclasts. Our findings suggest that TRPV-5 contributes to maintaining the homeostasis of the human skeleton via a negative feedback loop in RANKL-induced bone resorption.