RANKL from bone marrow adipose lineage cells promotes osteoclast formation and bone loss

Receptor activator of NF‐κB ligand (RANKL) is essential for osteoclast formation and bone remodeling. Nevertheless, the cellular source of RANKL for osteoclastogenesis has not been fully uncovered. Different from peripheral adipose tissue, bone marrow (BM) adipose lineage cells originate from bone marrow mesenchymal stromal cells (BMSCs). Here, we demonstrate that adiponectin promoter‐driven Cre expression (Adipoq^Cre ) can target bone marrow adipose lineage cells. We cross the Adipoq^Cre mice with rankl^fl/fl mice to conditionally delete RANKL from BM adipose lineage cells. Conditional deletion of RANKL increases cancellous bone mass of long bones in mice by reducing the formation of trabecular osteoclasts and inhibiting bone resorption but does not affect cortical bone thickness or resorption of calcified cartilage. Adipoq^Cre; rankl^fl/fl mice exhibit resistance to estrogen deficiency and rosiglitazone (ROS)‐induced trabecular bone loss but show bone loss induced by unloading. BM adipose lineage cells therefore represent an essential source of RANKL for the formation of trabecula osteoclasts and resorption of cancellous bone during remodeling under physiological and pathological conditions. Targeting bone marrow adiposity is a promising way of preventing pathological bone loss.     

Impaired osteoclast formation in bone marrow cultures of Fgf2 null mice in response to parathyroid hormone

Fibroblast growth factor (FGF)-2 and parathyroid hormone (PTH) are potent inducers of osteoclast (OCL) formation, and PTH increases FGF-2 mRNA and protein expression in osteoblasts. To elucidate the role of endogenous FGF-2 in PTH responses, we examined PTH-induced OCL formation in bone marrow cultures from wild type and mice with a disruption of the Fgf2 gene. FGF-2-induced OCL formation was similar in marrow culture from both genotypes. In contrast, PTH-stimulated OCL formation in bone marrow cultures or co-cultures of osteoblast-spleen cells from Fgf2-/mice was significantly impaired. PTH increased RANKL mRNA expression in osteoblasts cultures from both genotypes. After 6 days of treatment, osteoprotegerin protein in cell supernatants was 40-fold higher in vehicle-treated and 30-fold higher in PTH-treated co-cultures of osteoblast and spleen cells from Fgf2-/mice compared with Fgf2+/+ mice. However, a neutralizing antibody to osteoprotegerin did not rescue reduced OCL formation in response to PTH. Injection of PTH caused hypercalcemia in Fgf2+/+ but not Fgf2-/mice. We conclude that PTH stimulates OCL formation and bone resorption in mice in part by endogenous FGF-2 synthesis by osteoblasts. Because RANKL- and interleukin-11-induced OCL formation was also reduced in bone marrow cultures from Fgf2-/mice, we further conclude that endogenous FGF-2 is necessary for maximal OCL formation by multiple bone resorbing factors.     

IL-23 is critical for induction of arthritis, osteoclast formation, and maintenance of bone mass

The role of IL-23 in the development of arthritis and bone metabolism was studied using systemic IL-23 exposure in adult mice via hydrodynamic delivery of IL-23 minicircle DNA in vivo and in mice genetically deficient in IL-23. Systemic IL-23 exposure induced chronic arthritis, severe bone loss, and myelopoiesis in the bone marrow and spleen, which resulted in increased osteoclast differentiation and systemic bone loss. The effect of IL-23 was partly dependent on CD4(+) T cells, IL-17A, and TNF, but could not be reproduced by overexpression of IL-17A in vivo. A key role in the IL-23-induced arthritis was made by the expansion and activity of myeloid cells. Bone marrow macrophages derived from IL-23p19(-/-) mice showed a slower maturation into osteoclasts with reduced tartrate-resistant acid phosphatase-positive cells and dentine resorption capacity in in vitro osteoclastogenesis assays. This correlated with fewer multinucleated osteoclast-like cells and more trabecular bone volume and number in 26-wk-old male IL-23p19(-/-) mice compared with control animals. Collectively, our data suggest that systemic IL-23 exposure induces the expansion of a myeloid lineage osteoclast precursor, and targeting IL-23 pathway may combat inflammation-driven bone destruction as observed in rheumatoid arthritis and other autoimmune arthritides.     

SNX10 is required for osteoclast formation and resorption activity

RANKL-stimulation of osteoclast precursors results in up-regulation of genes involved in the process of differentiation and activation. In this report we describe the expression and functional characterization of Sorting Nexin 10 (snx10). Snx10 belongs to the sorting nexin (SNX) family, a diverse group of proteins with a common feature: the PX domain, which is involved in membrane trafficking and cargo sorting in endosomes. Snx10 is strongly up-regulated during RANKL-induced osteoclast differentiation in vitro and expressed in osteoclasts in vivo. qPCR analysis confirmed a significant increase in the expression of snx10 in in vitro-derived osteoclasts, as well as in femur and calvaria. Immunohistochemical analysis of mouse embryo sections showed expression in long bone, calvariae, and developing teeth. The expression was limited to cells that also expressed TRAP, demonstrating osteoclastic localization. Confocal immunofluorescence and subcellular fractionation analysis revealed Snx10 localization in the nucleus and in the endoplasmic reticulum (ER). To study a possible role for snx10 in osteoclast differentiation and function we silenced snx10 expression and found that snx10 silencing inhibited RANKL-induced osteoclast formation and osteoclast resorption on hydroxyapatite. Silencing also inhibited TRAP secretion. Taken together, these results confirm that snx10 is expressed in osteoclasts and is required for osteoclast differentiation and activity in vitro. Since inhibition of vesicular trafficking is essential for osteoclast formation and activity and SNX10 is involved in intracellular vesicular trafficking, these studies may identify a new candidate gene involved in the development of human bone diseases including osteoporosis.     

IL-6, RANKL, TNF-alpha/IL-1: interrelations in bone resorption pathophysiology

All osteogenic cells (osteoclasts, osteoblasts) contribute individually to bone remodeling. Their cellular interactions control their cellular activities and the bone remodeling intensity. These interactions can be established either through a cell-cell contact, involving molecules of the integrin family, or by the release of many polypeptidic factors and/or their soluble receptor chains. These factors can act directly on osteogenic cells and their precursors to control differentiation, formation and functions (matrix formation, mineralization, resorption...). Here, we present the involvement of three groups of cytokines which seem to be of particular importance in bone physiology: interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) (TNF-alpha)/IL-1, and the more recently known triad osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL). The interactions between these three groups are presented within the framework of bone resorption pathophysiology such as tumor associated osteolysis. The central role of the OPG/RANK/RANKL triad is pointed out.     

Interleukin-6 is not essential for bone turnover in hypothyroid mice

Interleukin-6 (IL-6) has been shown to be involved in the pathogenesis of several bone diseases characterized by an imbalance between bone resorption and formation. The aim of the study was to estimate serum markers of bone turnover: osteoclast-derived tartrate-resistant acid phosphatase form 5a (TRACP 5b) and osteocalcin in IL-6-deficient mice to assess the role of IL-6 in bone metabolism in hypothyroidism in mice. C57BL/6J (wild-type; WT) and C57BL/6J(IL6-/-Kopf) (IL-6 knock-out; IL6KO) mice randomly divided into 4 groups with 10 in each one: 1/ WT mice in hypothyroidism (WT-ht), 2/ WT controls, 3/ IL6KO mice with hypothyroidism (IL6KO-ht) and 4/ IL6KO controls. Experimental model of hypothyroidism was induced by intraperitoneal injection of propylthiouracyl. The serum levels of TRACP 5b and osteocalcin were determined by ELISA. Serum concentrations of TRACP 5b (median and interquartile ranges) were significantly decreased in both groups of mice with hypothyroidism: WT (3.2 (2.5-4.7) U/l) and IL6KO (2.6 (1.8-3.5) U/l) as compared to the respective controls. Similarly, serum osteocalcin levels were significantly reduced in both groups of mice in experimental hypothyroidism: WT (25.8 (23.0-28.2) ng/ml) and IL6KO (21.5(19.0-24.6) ng/ml) in comparison to the respective controls. There were no significant differences in bone turnover markers between IL6KO and WT mice both in hypothyroid and control animals. The results of the present study suggest that IL-6 does not play an important role in bone turnover in both euthyroid and hypothyroid mice.     

Calcium influx is not required for thyrotropin-releasing hormone stimulation of prolactin release from GH3 cells

TRH stimulation of prolactin release from GH3 cells is dependent on Ca2+; however, whether TRH-induced influx of extracellular Ca2+ is required for stimulated secretion remains controversial. We studied prolactin release from cells incubated in medium containing 110 mM K+ and 2 mM EGTA which abolished the electrical and Ca2+ concentration gradients that usually promote Ca2+ influx. TRH caused prolactin release and 45Ca2+ efflux from cells incubated under these conditions. In static incubations, TRH stimulated prolactin secretion from 11.4 +/- 1.2 to 19 +/- 1.8 ng/ml in control incubations and from 3.2 +/- 0.6 to 6.2 +/- 0.8 ng/ml from cells incubated in medium with 120 mM K+ and 2 mM EGTA. We conclude that Ca2+ influx is not required for TRH stimulation of prolactin release from GH3 cells.     

Specific antagonists of NMDA receptors prevent osteoclast sealing zone formation required for bone resorption

N-Methyl-d-aspartate (NMDA) glutamate receptors, widely distributed in the nervous system, have recently been identified in bone. They are expressed and are functional in osteoclasts. In the present work, we have studied the effects of specific antagonists of NMDA receptors on osteoclast activation and bone resorption. Using an in vitro assay of bone resorption, we showed that several antagonists of NMDA receptors binding to different sites of the receptor inhibit bone resorption. Osteoclast activation requires adhesion to the bone surface, cytoskeletal reorganization and survival. We demonstrated by autoradiography that the specific NMDA receptor channel blocker, MK 801, binds to osteoclasts. This antagonist had no effect on osteoclast attachment to bone and did not induce osteoclast apoptosis. In contrast, MK 801 rapidly decreased the percentage of osteoclasts with actin ring structures that are associated with actively resorbing osteoclasts. These results suggest that NMDA receptors expressed by osteoclasts may be involved in adhesion-induced formation of the sealing zone required for bone resorption.     

Bruton's tyrosine kinase is required for TLR2 and TLR4-induced TNF, but not IL-6, production

Bruton's tyrosine kinase (Btk), the gene mutated in the human immunodeficiency X-linked agammaglobulinemia, is activated by LPS and is required for LPS-induced TNF production. In this study, we have investigated the role of Btk both in signaling via another TLR (TLR2) and in the production of other proinflammatory cytokines such as IL-1beta, IL-6, and IL-8. Our data show that in X-linked agammaglobulinemia PBMCs, stimulation with TLR4 (LPS) or TLR2 (N-palmitoyl-S-[2, 3-bis(palmitoyloxy)-(2R)-propyl]-(R)-cysteine) ligands produces significantly less TNF and IL-1beta than in normal controls. In contrast, a lack of Btk has no impact on the production of IL-6, IL-8, or the anti-inflammatory cytokine, IL-10. Our previous data suggested that Btk lies within a p38-dependent pathway that stabilizes TNF mRNA. Accordingly, TaqMan quantitative PCR analysis of actinomycin D time courses presented in this work shows that overexpression of Btk is able to stabilize TNF, but not IL-6 mRNA. Furthermore, using the p38 inhibitor SB203580, we show that the TLR4-induced production of TNF, but not IL-6, requires the activity of p38 MAPK. These data provide evidence for a common requirement for Btk in TLR2- and TLR4-mediated induction of two important proinflammatory cytokines, TNF and IL-1beta, and reveal important differences in the TLR-mediated signals required for the production of IL-6, IL-8, and IL-10.     

Ellagic acid protects ovariectomy-induced bone loss in mice by inhibiting osteoclast differentiation and bone resorption

Osteoporosis is a devastating disease that features reduced bone quantity and microstructure, which causes fragility fracture and increases mortality, especially in the aged population. Due to the long-term side-effects of current drugs for osteoporosis, it is of importance to find other safe and effective medications. Ellagic acid (EA) is a phenolic compound found in nut galls, plant extracts, and fruits, and exhibits antioxidant and antineoplastic effects. Here, we showed that EA attenuated the formation and function of osteoclast dose-dependently. The underlying mechanism was further discovered by western blot, immunofluorescence assay, and luciferase assay, which elucidated that EA suppressed osteoclastogenesis and bone resorption mainly through attenuating receptor activator of nuclear factor-κB (NF-κB) ligand-induced NF-κB activation and extracellular signal-regulated kinase signaling pathways, accompanied by decreased protein expression of nuclear factor of activated T-cells calcineurin-dependent 1 and c-Fos. Moreover, EA inhibits osteoclast marker genes expression including Dc-stamp, Ctsk, Atp6v0d2, and Acp5. Intriguingly, we also found that EA treatment could significantly protect ovariectomy-induced bone loss in vivo. Conclusively, this study suggested that EA might have the therapeutic potentiality for preventing or treating osteoporosis.     

IL-23 inhibits osteoclastogenesis indirectly through lymphocytes and is required for the maintenance of bone mass in mice

IL-23 stimulates the differentiation and function of the Th17 subset of CD4(+) T cells and plays a critical role in chronic inflammation. The IL-23 receptor-encoding gene is also an inflammatory disease susceptibility gene. IL-23 shares a common subunit with IL-12, a T cell-dependent osteoclast formation inhibitor, and we found that IL-23 also dose-dependently inhibited osteoclastogenesis in a CD4(+) T lymphocyte-dependent manner. When sufficiently enriched, gammadelta T cells also mediated IL-23 inhibition. Like IL-12, IL-23 acted synergistically with IL-18 to block osteoclastogenesis but, unlike IL-12, IL-23 action depended on T cell GM-CSF production. IL-23 did not mediate IL-12 action although IL-12 induced its expression. Male mice lacking IL-23 (IL-23p19(-/-)) had approximately 30% lower bone mineral density and tibial trabecular bone mass (bone volume (BV)/total volume (TV)) than wild-type littermates at 12 wk and 40% lower BV/TV at 26 wk of age; male heterozygotes also had lower bone mass. Female IL-23p19(-/-) mice also had reduced BV/TV. IL-23p19(-/-) mice had no detectable osteoclast defect in trabecular bone but IL-23p19(-/-) had thinner growth plate hypertrophic and primary spongiosa zones (and, in females, less cartilage remnants) compared with wild type. This suggests increased osteoclast action at and below the growth plate, leading to reduced amounts of mature trabecular bone. Thus, IL-23 inhibits osteoclast formation indirectly via T cells in vitro. Under nonpathological conditions (unlike inflammatory conditions), IL-23 favors higher bone mass in long bones by limiting resorption of immature bone forming below the growth plate.     

IL-23 is required for neutrophil homeostasis in normal and neutrophilic mice

IL-23 is secreted by macrophages and dendritic cells in response to microbial products and inflammatory cytokines. IL-23 is a heterodimer composed of the unique IL-23p19 subunit linked to the common p40 subunit that it shares with IL-12. IL-23 is implicated in autoimmune diseases, where it supports the expansion of IL-17A-producing CD4+ Th17 cells. IL-23 also regulates granulopoiesis in a neutrostat regulatory feedback loop through IL-17A-producing neutrophil regulatory (Tn) cells, most of which express gammadelta TCR. This homeostatic system is disrupted in mice lacking adhesion molecules like beta2-integrins (Itgb2-/-) which have defective neutrophil trafficking and neutrophilia. To test the role of IL-23 in the homeostatic regulation of circulating neutrophil numbers, we measured blood neutrophil numbers in p40-deficient (IL12b-/-) mice and found them reduced compared with wild-type mice. IL12b-/-Itgb2-/- mice, lacking beta2-integrins, IL-12, and IL-23 showed significantly blunted neutrophilia compared with Itgb2-/- mice. Treatment of both IL12b-/- and IL12b-/-Itgb2-/- mice with IL-23, but not IL-12, restored circulating neutrophil counts. Serum levels of IL-17A were readily detectable in Itgb2-/- mice, but not in IL12b-/-Itgb2-/- mice, suggesting that IL-17A production is reduced when IL-23 is absent. Similarly, tissue mRNA expression of IL-17A was reduced in IL12b-/-Itgb2-/-mice compared with Itgb2-/- controls. The total number of CD3+ IL-17A-producing Tn cells were significantly reduced in the spleen and lamina propria of IL12b-/-Itgb2-/- mice, with the largest reduction found in gammadelta+ T cells. Our results suggest a prominent role of IL-23 in the regulation of granulopoiesis and the prevalence of IL-17A-producing Tn cells.     

Dietary emu oil supplementation suppresses 5-fluorouracil chemotherapy-induced inflammation, osteoclast formation, and bone loss

Cancer chemotherapy can cause osteopenia or osteoporosis, and yet the underlying mechanisms remain unclear, and currently, no preventative treatments are available. This study investigated damaging effects of 5-fluorouracil (5-FU) on histological, cellular, and molecular changes in the tibial metaphysis and potential protective benefits of emu oil (EO), which is known to possess a potent anti-inflammatory property. Female dark agouti rats were gavaged orally with EO or water (1 ml·day(-1)·rat(-1)) for 1 wk before a single ip injection of 5-FU (150 mg/kg) or saline (Sal) was given. The treatment groups were H(2)O + Sal, H(2)O + 5-FU, EO + 5-FU, and EO + Sal. Oral gavage was given throughout the whole period up to 1 day before euthanasia (days 3, 4, and 5 post-5-FU). Histological analysis showed that H(2)O + 5-FU significantly reduced heights of primary spongiosa on days 3 and 5 and trabecular bone volume of secondary spongiosa on days 3 and 4. It reduced density of osteoblasts slightly and caused an increase in the density of osteoclasts on trabecular bone surface on day 4. EO supplementation prevented reduction of osteoblasts and induction of osteoclasts and bone loss caused by 5-FU. Gene expression studies confirmed an inhibitory effect of EO on osteoclasts since it suppressed 5-FU-induced expression of proinflammatory and osteoclastogenic cytokine TNFα, osteoclast marker receptor activator of nuclear factor-κB, and osteoclast-associated receptor. Therefore, this study demonstrated that EO can counter 5-FU chemotherapy-induced inflammation in bone, preserve osteoblasts, suppress osteoclast formation, and potentially be useful in preventing 5-FU chemotherapy-induced bone loss.     

IL-22 is required for imiquimod-induced psoriasiform skin inflammation in mice

Psoriasis is a common chronic autoimmune skin disease of unknown cause that involves dysregulated interplay between immune cells and keratinocytes. IL-22 is a cytokine produced by the TH1, TH17, and TH22 subsets that are functionally implicated in the psoriatic pathology. We assessed the role of IL-22 in a mouse model where psoriasiform skin inflammation is triggered by topical application of the TLR7/8 agonist imiquimod. At the macroscopic level, scaly skin lesions induced by daily applications of imiquimod in wild-type mice were almost totally absent in IL-22-deficient mice or in mice treated with a blocking anti-IL-22 Ab. At the microscopic level, IL-22-deficient mice showed a dramatic decrease in the development of pustules and a partial decrease in acanthosis. At the molecular level, the absence or inhibition of IL-22 strongly decreased the expression of chemotactic factors such as CCL3 and CXCL3 and of biomarkers such as S100A8, S100A7, and keratin 14, which reflect the antimicrobial and hyperproliferative responses of keratinocytes. IL-22 also played a major role in neutrophil infiltration after imiquimod treatment. IL-23 was required for IL-22 production, and γδ TCR lymphocytes represented the major source of IL-22 in lymph nodes from imiquimod-treated mice. However, T cells were not absolutely required for IL-22 production because imiquimod-induced IL-22 expression in the skin is still preserved in Rag2(-/-) mice. Taken together, our data show that IL-22 is required for psoriasis-like lesions in the mouse imiquimod model and is produced by both T cells and innate immune cells.     

Hypoxia is a major stimulator of osteoclast formation and bone resorption

Hypoxia is known to act as a general stimulator of cells derived from marrow precursors. We investigated the effect of oxygen tension on the formation and function of osteoclasts, the cells responsible for bore resorption, which are of promonocytic origin. Using 7- and 13-day cultures of mouse marrow cells on ivory discs, we found that reducing oxygen tension from the ambient atmospheric level of 20% by increasing the proportion of nitrogen caused progressive increases in the formation of multinucleated osteoclasts and resorption pits. Peak effects occurred in 2% oxygen, where stimulations of resorption up to 21-fold were measured. Significant stimulations of osteoclast formation and resorption were observed even in severely hypoxic cultures gassed with 0.2% oxygen. Short-term cultures of cells disaggregated from rat bones indicated that hypoxia did not alter the resorptive activity of mature osteoclasts, but reduced their survival or adherence. In 3-day organ cultures of mouse calvarial bones, exposure to 2% oxygen resulted in maximal, fivefold stimulation of osteoclast-mediated calcium release, an effect equivalent to that of prostaglandin E(2) (PGE(2)), a reference osteolytic agent. Hypoxia also caused a moderate acidosis in calvarial cultures, presumably as a result of increased anaerobic metabolism; this observation is significant because osteoclast activation is dependent on extracellular acidification. Our experiments reveal a previously-overlooked mechanism of considerable potential importance for the regulation of bone destruction. These findings may help explain the bone loss associated with a wide range of pathological states involving local or systemic hypoxia, and emphasize the importance of the vasculature in bone.