Regulation of osteoclastogenesis by integrated signals from toll-like receptors.

A variety of pathogen-derived molecules have been shown to cause bone loss by enhancing osteoclast differentiation through activation of toll-like receptors (TLRs). The pathogen-derived molecules (TLR-ligands) modulate osteoclastogenesis in a complex manner: inhibition of the osteoclast differentiation factor RANKL in early precursors and osteoclastogenesis stimulation in RANKL-primed cells. Since organisms may be challenged by several TLR ligands at a time, we investigated osteoclastogenesis modulation by simultaneous challenge with different TLR ligands. As an example we used ligands for TLR3 (Synthetic double stranded RNA [dsRNA], polyinosinic-polycytidylic acid [poly(I:C)] mimicking viral dsRNA), TLR4 (lipopolysaccharide [LPS], found in the outer membrane of Gram-negative bacteria) and TLR9 (Synthetic oligodeoxynucleotide mimicking bacterial DNA [CpG-ODN]). In osteoclastogenesis-inhibition, synergy between LPS and CpG-ODN or LPS and poly(I:C) while in stimulation, synergy between LPS and CpG-ODN or CpG-ODN and poly(I:C) were observed. Modulation of molecules involved in osteoclastogenesis (c-Fos, M-CSF receptors [M-CSFR], TNF-α, IL-6, and IL-12 and the three TLRs tested) was examined. The results indicate that M-CSFR plays a role only in the inhibitory effect while c-Fos plays a role in the two effects. TLR3 and TLR9 levels were increased by the TLRs ligands, suggesting that this may be part of the mechanism leading to the synergy. While TLRs activation in RANKL-primed cells, increasing osteoclastogenesis, explains pathogen-induced bone loss, activation of TLRs in early cells inhibiting osteoclastogenesis could attenuate excessive resorption, and promote differentiation of common precursor cells into inflammatory cells. The synergism between TLR ligands enables the individual to initiate response at a lower level of pathogen.

An SNX10 mutation causes malignant osteopetrosis of infancy.

BACKGROUND: Osteopetrosis is a life-threatening, rare disorder typically resulting from osteoclast dysfunction and infrequently from failure to commitment to osteoclast lineage. Patients commonly present in infancy with macrocephaly, feeding difficulties, evolving blindness and deafness, and bone marrow failure. In ∼70% of the patients there is a molecularly defined failure to maintain an acid pH at the osteoclast-bone interface (the ruffled border) which is necessary for the bone resorptive activity. METHODS AND RESULTS: In eight patients with infantile osteopetrosis which could be cured by bone marrow transplantation, the study identified by homozygosity mapping in distantly related consanguineous pedigrees a missense mutation in a highly conserved residue in the SNX10 gene. The mutation segregated with the disease in the families and was carried by one of 211 anonymous individuals of the same ethnicity. In the patients' osteoclasts, the mutant SNX10 protein was abnormally abundant and its distribution altered. The patients' osteoclasts were fewer and smaller than control cells, their resorptive capacity was markedly deranged, and the endosomal pathway was perturbed as evidenced by the distribution of internalised dextran. CONCLUSIONS: SNX10 was recently shown to interact with vacuolar type H(+)-ATPase (V-ATPase) which pumps protons at the osteoclast-bone interface. Mutations in TCIRG1, the gene encoding a subunit of the V-ATPase complex, account for the majority of cases of osteopetrosis. It is speculated that SNX10 is responsible for the vesicular sorting of V-ATPase from Golgi or for its targeting to the ruffled border. A mutation in SNX10 may therefore result in 'secondary V-ATPase deficiency' with a failure to acidify the resorption lacuna. Determination of the sequence of the SNX10 gene is warranted in molecularly undefined patients with recessive 'pure' osteopetrosis of infancy.

Toll-like receptor 9 ligand blocks osteoclast differentiation through induction of phosphatase.

UNLABELLED: CpG-ODN, in addition to stimulation of osteoclastogenic signals in early osteoclast precursors, also induces phosphatase, shifting the pattern of ERK phosphorylation from sustained to transient. This shift results in the degradation of c-fos, an essential molecule for osteoclast differentiation. Therefore, CpG-ODN blocks osteoclast differentiation. INTRODUCTION: Activation of either Toll-like receptor 9 (TLR9) or RANK induces similar responses in osteoclast precursors. Paradoxically, activation of TLR9 results in inhibition of RANKL-induced osteoclastogenesis. MATERIALS AND METHODS: We used bone marrow-derived osteoclast precursors. Analyses of signaling molecules phosphorylation were performed using Western blotting. Different levels of gene expression analyses were performed using RT-PCR, Northern, and run-on analyses (for RNA), and EMSA, Western, and pulse-chase experiments (for protein). Phosphatase activity was measured spectrophotometrically. RESULTS: We found that RANKL and TLR9 ligand, oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG-ODN), induce sustained and transient extracellular signal-regulated kinase (ERK) phosphorylation, respectively. Furthermore, together they induce a transient phosphorylation of ERK. The duration of ERK phosphorylation is a key factor in determining induction of c-fos, a protein critical for osteoclastogenesis. Indeed, we found that CpG-ODN does not induce c-fos and inhibits its induction by RANKL by enhancing c-fos mRNA and protein degradation. Our observation that CpG-ODN, but not RANKL, induces the expression of the phosphatase PP2A suggests that CpG-ODN exerts its inhibitory activity by induction of ERK dephosphorylation. Moreover, together with the phosphatase inhibitor okadaic acid, CpG-ODN induces sustained ERK phosphorylation and c-fos expression. CONCLUSIONS: Our findings suggest that the increased rate of c-fos degradation by the TLR9 ligand mediates the inhibition of RANKL-induced osteoclast differentiation. The TLR9 ligand, through induction of dephosphorylation, prevents the sustained ERK phosphorylation needed for maintaining high c-fos levels that are essential for osteoclast differentiation.

Differential contribution of osteoclast- and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis.

UNLABELLED: CpG-ODNs modulate osteoclast differentiation through Toll-like receptor 9 (TLR9). Using TLR9-deficient mice, we found that activation of TLR9 on both osteoclast precursors and osteoblasts mediate the osteoclastogenic effect of CpG-ODN. Osteoclastic TLR9 is more important for this activity. INTRODUCTION: Bacterial infections cause pathological bone loss by accelerating differentiation and activation of the osteoclast. A variety of bacteria-derived molecules have been shown to enhance osteoclast differentiation through activation of Toll-like receptors (TLRs). We have shown that CpG-oligodeoxynucleotides (CpG-ODNs), mimicking bacterial DNA and exerting their cellular activities through TLR9, modulate osteoclast differentiation in a complex manner: the ODNs inhibit the activity of the physiological osteoclast differentiation factor RANKL in early osteoclast precursors (OCPs) but markedly stimulate osteoclastogenesis in cells primed by RANKL. MATERIALS AND METHODS: Osteoclast precursors and osteoblasts from TLR9-deficient (TLR9-/-) and wildtype (TLR9+/+) mice were used for in vitro analyses of osteoclast differentiation and modulation of signal transduction and gene expression. RESULTS: As expected CpG-ODN did not exert any activity in cells derived from TLR9-/-mice; these cells, however, responded in a normal manner to other stimuli. Using bone marrow/osteoblasts co-cultures from all possible combinations of TLR9-/- and TLR9+/+ mice-derived cells, we showed that TLR9 in the two lineages is required for CpG-ODN induction of osteoclastogenesis. CONCLUSIONS: CpG-ODN modulates osteoclastogenesis in a TLR9-dependent manner. Activation of TLR9 in bone marrow-derived osteoclasts precursors is more crucial to induction of osteoclastogenesis than activation of the osteoblastic TLR9.

CpG oligonucleotides: novel regulators of osteoclast differentiation.

The macrophage capability to recognize bacterial DNA is mimicked by oligodeoxynucleotides containing unmethylated CG dinucleotides ('CpG' motifs) in specific sequence contexts (CpG ODN). CpG ODN stimulates NF-kappaB activation in murine macrophages. In light of the pivotal role played by NF-kappaB in osteoclast differentiation, we examined the ability of CpG ODN to modulate osteoclastogenesis. CpG ODN alone induced TRAP-positive cells in bone marrow macrophage (BMM) cultures, but not multinucleation or calcitonin receptor expression. CpG ODN inhibited RANKL-induced osteoclastogenesis when present from the beginning of BMM culture, but strongly increased RANKL-induced osteoclastogenesis in RANKL-pretreated BMMs. CpG ODN enhanced the expression of interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha). Antibodies to TNF-alpha and the TNF type 1 receptor, but not the addition of IL-1 receptor antagonist, blocked CpG ODN-induced osteoclastogenesis in RANKL-pretreated cultures. On the other hand, CpG ODN reduced expression of the M-CSF receptor, which is critical during the initiation of osteoclast differentiation. These results suggest that CpG ODN, via the induction of TNF-alpha, support osteoclastogenesis in cells that are committed to the osteoclast differentiation pathway but, due to down-modulation of M-CSF receptor, inhibit early steps of osteoclast differentiation. Thus, CpG ODN represents a potential therapeutic tool for treating bone diseases.

Dual modulation of osteoclast differentiation by lipopolysaccharide.

Lipopolysaccharide (LPS) modulates bone resorption by augmentation of osteoclastogenesis. It increases in osteoblasts the production of RANKL, interleukin (IL)-1, prostaglandin E2 (PGE2), and TNF-alpha, each known to induce osteoclast activity, viability, and differentiation. We examined the role of direct interactions of LPS with osteoclast precursors in promoting their differentiation. To this end, we have used bone marrow mononuclear cell preparations in the absence of osteoblasts or stromal cells. We found that LPS does not induce osteoclast differentiation in these cells. Moreover, the inclusion of LPS blocked the osteoclastogenic activity of RANKL. However, LPS is a potent inducer of osteoclastogenesis in RANKL-pretreated cells, even if present in the absence of exogenous RANKL. Osteoprotegerin (OPG), does not affect the stimulatory phase of LPS modulation of osteoclastogenesis, ruling out involvement of endogenous RANKL. LPS induces the expression of TNF-a and IL-1beta in osteoclast precursors, regardless if they were or were not pretreated with RANKL. These two cytokines induced osteoclast differentiation in RANKL-pretreated cells. To examine if these cytokines mediate LPS effect in an autocrine mechanism, we measured the effect of their neutralization on LPS osteoclastogenic activity. Although neutralization of IL-1beta did not affect LPS activity, a marked inhibition was observed when TNF-alpha was neutralized. However, TNF-a expression was increased also in conditions in which LPS inhibited RANKL osteoclastogenic activity. We found that LPS reduces the expression of RANK and macrophage colony-stimulating factor (M-CSF) receptor. In summary, LPS impacts on osteoclastogenesis also via its interactions with the precursor cells. LPS inhibits RANKL activity by reducing the expression of RANK and M-CSF receptor and stimulates osteoclastogenesis in RANKL-pretreated cells via TNF-alpha.

Taking a toll on the bones: regulation of bone metabolism by innate immune regulators.

The interplay between the immune system and bone metabolism has been recognized as important for both of these systems. Various factors produced and released during immune responses markedly affect bone cells and bone metabolism. Meanwhile, niches for lymphocytes in bone also play an important role in the biology of these cells. Osteoimmunology, a new area of research focusing on associations between the immune and bone systems, is based on the concept that deeper investigation of the relationships between these systems will enhance our understanding of their biology and contribute to the discovery of novel therapeutic approaches for diseases of the two systems. Toll-like receptors (TLRs), the focus of this review, sense pathogen-derived molecules and initiate the inflammatory reactions of innate immune cells. TLRs are also expressed in bone cells, and their activation affects osteoclast differentiation and activity in a complex manner: TLR activation in early osteoclast precursors blocks the differentiation of those cells, while in cells that have already started their osteoclastic differentiation, it stimulates this process and increases the survival rates of mature osteoclasts (OCs). Activation of TLRs in osteoblasts (OBs) induces the production of osteoclastogenic cytokines, such as RANKL and TNF-alpha, thereby contributing to TLR ligand-induced osteoclastogenesis. These processes are the reason for the bone loss observed in variety of infectious diseases. The inhibition of osteoclastogenesis by TLR activation in early precursor cells may play a role in reducing the excessive bone loss caused by pathogenic infection and shifting the balance between the bone and immune systems during infection to recruit immune cells.

The osteoclast: a multinucleated, hematopoietic-origin, bone-resorbing osteoimmune cell.

Osteoclasts are multinucleated cells that derive from hematopoietic progenitors in the bone marrow which also give rise to monocytes in peripheral blood, and to the various types of tissue macrophages. Osteoclasts are formed by the fusion of precursor cells. They function in bone resorption and are therefore critical for normal skeletal development (growth and modeling), for the maintenance of its integrity throughout life, and for calcium metabolism (remodeling). To resorb bone, the osteoclasts attach to the bone matrix, their cytoskeleton reorganizes, and they assume polarized morphology and form ruffled borders to secrete acid and collagenolytic enzymes and a sealing zone to isolate the resorption site. Identification of the osteoclastogenesis inducer, the receptor activator of nuclear factor-kappaB ligand (RANKL), its cognate receptor RANK, and its decoy receptor osteoprotegerin (OPG), has contributed enormously to the dramatic advance in our understanding of the molecular mechanisms involved in osteoclast differentiation and activity. This explosion in osteoclast biology is reflected by the large number of reviews which appeared during the last decade. Here I will summarize the "classical" issues (origin, differentiation, and activity) in a general manner, and will discuss an untouched issue (multinucleation) and a relatively novel aspect of osteoclast biology (osteoimmunology).

Interleukin (IL)-12 mediates the anti-osteoclastogenic activity of CpG-oligodeoxynucleotides.

Bacterial DNA activates the innate immune system via interactions with Toll-like receptor 9 (TLR9). This receptor recognizes CpG-oligodeoxynucleotides (CpG-ODNs) mimicking the CpG dinucleotides in certain sequence contexts characterizing this DNA. Most studies have shown increased osteoclast differentiation by TLR ligands. We found that activation of TLRs (specifically TLR4 and TLR9) in early osteoclast precursors results in inhibition of receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation. Our objective is to identify the mechanism leading to this inhibitory effect of a TLR ligand. Since both RANKL-RANK and CpG-ODN-TLR9 interactions result in NF-kappaB activation, p38 and ERK phosphorylation, and TNF-alpha synthesis (all implicated in osteoclastogenesis), we hypothesized that CpG-ODN (but not RANKL) in addition induces the synthesis of an anti-osteoclastogenic factor. Control osteoclast precursors, and cells treated with RANKL, CpG-ODN, or their combination were studied using DNA arrays (GEArray Q Series Mouse NF-kappaB Signaling Pathway Gene Array, MM-016, SuperArray). We found a marked increase in the mRNA levels of the osteoclastogenesis inhibitor interleukin-12 (IL-12) in osteoclast precursors treated with CpG-ODN and CpG-ODN + RANKL. Northern and Western analyses, together with ELISA, confirmed the DNA array studies. In correlation with these findings, IL-12 inhibited RANKL-induced osteoclast differentiation and specific anti-IL-12-antibodies inhibited the anti-osteoclastogenic effect of CpG-ODN. In conclusion, activation of TLR9 by its ligand, CpG-ODN, results in synthesis and release of IL-12 opposing RANKL-induced osteoclast differentiation.

Involvement of aquaporin 9 in osteoclast differentiation.

Aquaporins (water channels) selectively enhance water permeability of membranes. Since osteoclast differentiation includes a dramatic increase in cell volume, we hypothesize that aquaporin(s) is/are critical for the formation of the multinucleated osteoclast from its mononuclear precursor. Our studies employ two cell models, bone marrow macrophages (BMMs) and the murine macrophage-like cell line, RAW264.7, as osteoclast precursors. Receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) and macrophage-colony-stimulating factor or RANKL alone were used to induce osteoclast differentiation in BMMs or RAW264.7 cells, respectively. We first used qualitative reverse transcription (RT)-PCR to examine which of the aquaporins are expressed in osteoclasts and in their precursor cells. Out of the 10 aquaporins examined, only aquaporin 9 (AQP9) was expressed in osteoclast-lineage cells. AQP9 has unique aqueous pore properties mediating the passage of a wide variety of non-charged solutes in addition to water. Western analyses using specific antibodies revealed a higher AQP9 level in RANKL-treated than in untreated cells. Quantitative real-time RT-PCR analyses also demonstrated higher AQP9 mRNA levels in RANKL-treated cells. Finally, we examined the effect of phloretin, an AQP9 inhibitor, on RANKL-induced osteoclast differentiation. Cells were incubated with RANKL for 5 days, and phloretin was added for the last 2 days, when most fusion occurs. A dramatic reduction in osteoclast size and in the number of nuclei per osteoclast was observed in cultures containing phloretin. The inhibitor did not have a significant effect on the number and size of mononuclear phagocytes in cultures not treated with RANKL. Our results suggest a role for AQP9 in osteoclast differentiation, specifically in the fusion process.

Organization of transcriptional regulatory machinery in osteoclast nuclei: compartmentalization of Runx1.

The osteoclast is a highly polarized multinucleated cell that resorbs bone. Using high resolution immunofluorescence microscopy, we demonstrated that all nuclei of an osteoclast are transcriptionally active. Each nucleus within the osteoclast contains punctately organized microenvironments where regulatory complexes that support transcriptional and post-transcriptional control reside. Functional equivalency of osteoclast nuclei is reflected by similar representation of regulatory proteins that support ribosomal RNA synthesis (nucleolin), mRNA transcription (RNA polymerase II, bromouridine triphosphate), processing of gene transcripts (SC35), signal transduction (NF-kappaB), and phenotypic gene expression (Runx1). Our results establish that gene regulatory machinery is architecturally associated and compartmentalized within intranuclear microenvironments of the multiple nuclei of osteoclasts to support physiologically responsive modifications in cellular structural and functional properties.

Modulation of TNF-alpha expression in bone marrow macrophages: involvement of vitamin D response element.

The calcium-regulating hormone, 1,25(OH)(2)D(3), induces tumor necrosis factor-alpha (TNF-alpha) synthesis and release from bone marrow macrophages (BMMs). To investigate the mechanism of this regulation, we have examined the effects of 1,25(OH)(2)D(3) on the cytokine message. 1,25(OH)(2)D(3) increased TNF-alpha mRNA abundance in a dose- and time-dependent manner. The combined treatment of BMMs with LPS and 1,25(OH)(2)D(3) resulted in a synergistic increase of TNF-alpha. The steroid also increased the expression of CD14 (LPS receptor). Vitamin D receptors (VDRs) mediate 1,25(OH)(2)D(3) genomic effects by forming homodimers or heterodimers with retinoic acid receptors (RARs) or retinoic X receptors (RXRs). The RXR ligand, 9-cis retinoic acid (9cRA), reduced TNF-alpha mRNA abundance in BMMs, but increased CD14 mRNA levels. 1,25(OH)(2)D(3) or LPS did not affect TNF-alpha transcript stability. 9cRA, however, caused TNF-alpha mRNA destabilization. Next, we searched for potential vitamin D response elements (VDREs) in the promoter region (1.2 kb) of the TNF-alpha gene, and identified six such sequences. Using electrophoresis mobility shift assay (EMSA) we identified one of those sequences (-1008 to -994) as a likely candidate to be a VDRE (tnfVDRE). The binding of tnfVDRE to BMM-derived nuclear extract was increased following cell treatment with 1,25(OH)(2)D(3). No induction was observed with 9cRA treatment, but the retinoid enhanced the activity of 1,25(OH)(2)D(3) when added together. Previously characterized VDREs (mouse osteopontin and rat osteocalcin) competed effectively with tnfVDRE, demonstrating the nature of the TNF-alpha-derived sequence as a VDRE. We observed super-shift and block-shift of the complex in the presence of either anti-VDR or anti-RXR antibodies. Our data suggest that 1,25(OH)(2)D(3) increases TNF-alpha transcript abundance in BMMs via a transcriptional mechanism; 9cRA decreases TNF-alpha mRNA by destabilizing the transcript, and possibly also by forming transcriptionally inactive complex with 1,25(OH)(2)D(3) on the tnfVDRE. The receptor complex interacting with tnfVDRE found in the promoter of the cytokine gene is probably composed of VDR-RXR heterodimer.

CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9.

Regulation of osteoclastogenesis by lipopolysaccharide (LPS) is mediated via its interactions with toll-like receptor 4 (TLR4) on both osteoclast- and osteoblast-lineage cells. We have recently demonstrated that CpG oligodeoxynucleotides (CpG ODNs), known to mimic bacterial DNA, modulate osteoclastogenesis via interactions with osteoclast precursors. In the present study we characterize the interactions of CpG ODNs with osteoblasts, in comparison with LPS. We find that, similar to LPS, CpG ODNs modulate osteoclastogenesis in bone marrow cell/osteoblast co-cultures, although in a somewhat different pattern. Osteoblasts express receptors for both LPS and CpG ODN (TLR4 and TLR9, respectively). The osteoblastic TLR9 transmits signals into the cell as demonstrated by NFkappaB activation as well as by extracellular-regulated kinase (ERK) and p38 phosphorylation. Similar to LPS, CpG ODN increases in osteoblasts the expression of tumor necrosis factor (TNF)-alpha and macrophage-colony stimulating factor (M-CSF). The two TLR ligands do not affect osteoprotegerin expression in osteoblasts. CpG ODN does not significantly affect receptor activator of NFkappaB ligand (RANKL) expression, in contrast to LPS, which induces the expression of this molecule. In the co-cultures CpG ODN induces RANKL expression in osteoblasts as a result of the more efficient TNF-alpha induction. CpG ODN activity (modulation of osteoclastogenesis, gene expression, ERK and p38 phosphorylation, and nuclear translocation of NFkappaB) is specific, because the control oligodeoxynucleotide, not containing CpG, is inactive. Furthermore, these effects (unlike the LPS effects) are inhibited by chloroquine, suggesting a requirement for endosomal maturation/acidification, the classic CpG ODN mode of action. We conclude that CpG ODN, upon TLR9 ligation, induces osteoblasts osteoclastogenic activity.

Toll-like receptor 9 regulates tumor necrosis factor-alpha expression by different mechanisms. Implications for osteoclastogenesis.

CpG oligodeoxynucleotides (CpG-ODNs), mimicking bacterial DNA, stimulate osteoclastogenesis via Toll-like receptor 9 (TLR9) in receptor activator of NF-kappa B ligand (RANKL)-primed osteoclast precursors. This activity is mediated via tumor necrosis factor (TNF)-alpha induction by CpG-ODN. To further reveal the role of the cytokine in TLR9-mediated osteoclastogenesis, we compared the ability of CpG-ODN to induce osteoclastogenesis in two murine strains, BALB/c and C57BL/6, expressing different TNF-alpha alleles. The induction of osteoclastogenesis and TNF-alpha release by CpG-ODN was by far more noticeable in BALB/c-derived than in C57BL/6-derived osteoclast precursors. Unexpectedly, as revealed by Northern analysis, CpG-ODN induction of TNF-alpha mRNA increase was more efficient in C57BL/6-derived cells. The cytokine transcript abundance was increased due to both increased message stability and rate of transcription. The difference between the two cell types was the result of a higher transcription rate in CpG-ODN-induced C57BL/6-derived cells caused by a single nucleotide polymorphism in kappa B2a site within the TNF-alpha promoter sequence. CpG-ODN enhanced the rate of the cytokine translation in BALB/c-derived cells. Thus, CpG-ODN modulated both transcription and translation of TNF-alpha. The induction of transcription was more evident in C57BL/6-derived cells, while the induction of translation took place only in BALB/c-derived osteoclast precursors. Altogether the cytokine was induced to a larger extent in BALB/c-derived osteoclast precursors, consistent with the increased CpG-ODN osteoclastogenic effect in these cells.