Vitamin A enhanced periosteal osteoclastogenesis is associated with increased number of tissue-derived macrophages/osteoclast progenitors.

A deleterious effect of elevated levels of vitamin A on bone health has been reported in clinical studies. Mechanistic studies in rodents have shown that numbers of periosteal osteoclasts are increased, while endocortical osteoclasts are simultaneously decreased by vitamin A treatment. The present study investigated the in vitro and in vivo effect of all-trans retinoic acid (ATRA), the active metabolite of vitamin A, on periosteal osteoclast progenitors. Mouse calvarial bone cells were cultured in media containing ATRA, with or without the osteoclastogenic cytokine receptor activator of nuclear factor kappa B-ligand (RANKL), on plastic dishes or bone discs. Whereas ATRA did not stimulate osteoclast formation alone, the compound robustly potentiated the formation of RANKL-induced bone resorbing osteoclasts. This effect was due to stimulation by ATRA (half-maximal stimulation ∼3 nM) on the numbers of macrophages/osteoclast progenitors in the bone cell cultures, as assessed by mRNA and protein expression of several macrophage and osteoclast progenitor cell markers, such as macrophage colony-stimulating factor receptor, receptor activator of nuclear factor kappa B, F4/80, and CD11b, as well as by flow cytometry (FACS) analysis of CD11b+/F480+/Gr1- cells. The stimulation of macrophage numbers in the periosteal cell cultures was not mediated by increased macrophage colony-stimulating factor or interleukin-34. In contrast, ATRA did not enhance macrophages in bone marrow cell cultures. Importantly, ATRA treatment upregulated the mRNA expression of several macrophage-related genes in the periosteum of tibia in adult mice. These observations demonstrate a novel mechanism by which vitamin A enhances osteoclast formation specifically on periosteal surfaces.

The novel cytotoxic polybisphosphonate osteodex decreases bone resorption by enhancing cell death of mature osteoclasts without affecting osteoclastogenesis of RANKL-stimulated mouse bone marrow macrophages.

It has previously been demonstrated that the polybisphosphonate osteodex (ODX) inhibits bone resorption in organ-cultured mouse calvarial bone. In this study, we further investigate the effects by ODX on osteoclast differentiation, formation, and function in several different bone organ and cell cultures. Zoledronic acid (ZOL) was used for comparison. In retinoid-stimulated mouse calvarial organ cultures, ODX and ZOL significantly reduced the numbers of periosteal osteoclasts without affecting Tnfsf11 or Tnfrsf11b mRNA expression. ODX and ZOL also drastically reduced the numbers of osteoclasts in cell cultures isolated from the calvarial bone and in vitamin D3-stimulated mouse crude bone marrow cell cultures. These data suggest that ODX can inhibit osteoclast formation by inhibiting the differentiation of osteoclast progenitor cells or by directly targeting mature osteoclasts. We therefore assessed if osteoclast formation in purified bone marrow macrophage cultures stimulated by RANKL was inhibited by ODX and ZOL and found that the initial formation of mature osteoclasts was not affected, but that the bisphosphonates enhanced cell death of mature osteoclasts. In agreement with these findings, ODX and ZOL did not affect the mRNA expression of the osteoclastic genes Acp5 and Ctsk and the osteoclastogenic transcription factor Nfatc1. When bone marrow macrophages were incubated on bone slices, ODX and ZOL inhibited RANKL-stimulated bone resorption. In conclusion, ODX does not inhibit osteoclast formation but inhibits osteoclastic bone resorption by decreasing osteoclast numbers through enhanced cell death of mature osteoclasts.

Osteoprotective effect by interleukin-4 (IL-4) on lipoprotein-induced periodontitis.

Lipoproteins are immunostimulatory bacterial components suggested to participate in inflammation-induced bone loss in periodontal disease through stimulation of osteoclast differentiation. Toll-like receptor 2 activation by Pam2CSK4 (PAM2), known to mimic bacterial lipoproteins, was previously shown to enhance periodontal bone resorption in mice. The anti-inflammatory cytokine interleukin-4 (IL-4) is a known inhibitor of RANKL-induced bone resorption in vitro. Here, we have investigated whether IL-4 could decrease PAM2-induced periodontal bone loss and osteoclastogenesis in vivo. In a model of periodontitis induced by gingival injections of PAM2 in mice, concomitant injections of IL-4 reduced bone loss. Histologically, IL-4 reduced the recruitment of inflammatory cells and the formation of TRAP+ osteoclasts stimulated by PAM2. Mouse bone marrow macrophages (BMMs) and neonatal calvarial osteoblasts were used to assess the effect of IL-4 on PAM2-induced osteoclastogenesis in vitro. In RANKL-primed BMMs stimulated by PAM2 Nfatc1, Ctsk, and Acp5 gene expression was up-regulated and resulted in robust formation of TRAP+ multinucleated osteoclasts, effects which were impaired by IL-4. These effects were mediated by impairment in PAM2-induced c-fos expression. In primary calvarial osteoblast cultures, IL-4 decreased PAM2-induced Tnfsf11 (encoding RANKL) mRNA and enhanced Tnfrsf11b (encoding OPG) expression. Our data demonstrate that the osteoprotective effect by IL-4 on lipoprotein-induced periodontal disease occurs through the inhibition of osteoclastogenesis by three mechanisms, one by acting directly on osteoclast progenitors, another by acting indirectly through decreasing the expression of osteoclast-regulating cytokines in osteoblasts and a third by decreasing inflammation.

Estradiol and RSPO3 regulate vertebral trabecular bone mass independent of each other.

Osteoporosis is an age-dependent serious skeletal disease that leads to great suffering for the patient and high social costs, especially as the global population reaches higher age. Decreasing estrogen levels after menopause result in a substantial bone loss and increased fracture risk, whereas estrogen treatment improves bone mass in women. RSPO3, a secreted protein that modulates WNT signaling, increases trabecular bone mass and strength in the vertebrae of mice, and is associated with trabecular density and risk of distal forearm fractures in humans. The aim of the present study was to determine if RSPO3 is involved in the bone-sparing effect of estrogens. We first observed that estradiol (E2) treatment increases RSPO3 expression in bone of ovariectomized (OVX) mice, supporting a possible role of RSPO3 in the bone-sparing effect of estrogens. As RSPO3 is mainly expressed by osteoblasts in the bone, we used a mouse model devoid of osteoblast-derived RSPO3 (Runx2-creRspo3flox/flox mice) to determine if RSPO3 is required for the bone-sparing effect of E2 in OVX mice. We confirmed that osteoblast-specific RSPO3 inactivation results in a substantial reduction in trabecular bone mass and strength in the vertebrae. However, E2 increased vertebral trabecular bone mass and strength similarly in mice devoid of osteoblast-derived RSPO3 and control mice. Unexpectedly, osteoblast-derived RSPO3 was needed for the full estrogenic response on cortical bone thickness. In conclusion, although osteoblast-derived RSPO3 is a crucial regulator of vertebral trabecular bone, it is required for a full estrogenic effect on cortical, but not trabecular, bone in OVX mice. Thus, estradiol and RSPO3 regulate vertebral trabecular bone mass independent of each other.NEW & NOTEWORTHY Osteoblast-derived RSPO3 is known to be a crucial regulator of vertebral trabecular bone. Our new findings show that RSPO3 and estrogen regulate trabecular bone independent of each other, but that RSPO3 is necessary for a complete estrogenic effect on cortical bone.

Stimulation of Osteoclast Formation by Oncostatin M and the Role of WNT16 as a Negative Feedback Regulator.

Oncostatin M (OSM), which belongs to the IL-6 family of cytokines, is the most potent and effective stimulator of osteoclast formation in this family, as assessed by different in vitro assays. Osteoclastogenesis induced by the IL-6 type of cytokines is mediated by the induction and paracrine stimulation of the osteoclastogenic cytokine receptor activator of nuclear factor κ-B ligand (RANKL), expressed on osteoblast cell membranes and targeting the receptor activator of nuclear factor κ-B (RANK) on osteoclast progenitor cells. The potent effect of OSM on osteoclastogenesis is due to an unusually robust induction of RANKL in osteoblasts through the OSM receptor (OSMR), mediated by a JAK-STAT/MAPK signaling pathway and by unique recruitment of the adapter protein Shc1 to the OSMR. Gene deletion of Osmr in mice results in decreased numbers of osteoclasts and enhanced trabecular bone caused by increased trabecular thickness, indicating that OSM may play a role in physiological regulation of bone remodeling. However, increased amounts of OSM, either through administration of recombinant protein or of adenoviral vectors expressing Osm, results in enhanced bone mass due to increased bone formation without any clear sign of increased osteoclast numbers, a finding which can be reconciled by cell culture experiments demonstrating that OSM can induce osteoblast differentiation and stimulate mineralization of bone nodules in such cultures. Thus, in vitro studies and gene deletion experiments show that OSM is a stimulator of osteoclast formation, whereas administration of OSM to mice shows that OSM is not a strong stimulator of osteoclastogenesis in vivo when administered to adult animals. These observations could be explained by our recent finding showing that OSM is a potent stimulator of the osteoclastogenesis inhibitor WNT16, acting in a negative feedback loop to reduce OSM-induced osteoclast formation.

Osteocyte- and late osteoblast-derived NOTUM reduces cortical bone mass in mice.

Osteoporosis is a common skeletal disease, with increased risk of fractures. Currently available osteoporosis treatments reduce the risk of vertebral fractures, mainly dependent on trabecular bone, whereas the effect on nonvertebral fractures, mainly dependent on cortical bone, is less pronounced. WNT signaling is a crucial regulator of bone homeostasis, and the activity of WNTs is inhibited by NOTUM, a secreted WNT lipase. We previously demonstrated that conditional inactivation of NOTUM in all osteoblast lineage cells increases the cortical but not the trabecular bone mass. The aim of the present study was to determine if NOTUM increasing cortical bone is derived from osteoblast precursors/early osteoblasts or from osteocytes/late osteoblasts. First, we demonstrated Notum mRNA expression in Dmp1-expressing osteocytes and late osteoblasts in cortical bone using in situ hybridization. We then developed a mouse model with inactivation of NOTUM in Dmp1-expressing osteocytes and late osteoblasts (Dmp1-creNotumflox/flox mice). We observed that the Dmp1-creNotumflox/flox mice displayed a substantial reduction of Notum mRNA in cortical bone, resulting in increased cortical bone mass and decreased cortical porosity in femur but no change in trabecular bone volume fraction in femur or in the lumbar vertebrae L5 in Dmp1-creNotumflox/flox mice as compared with control mice. In conclusion, osteocytes and late osteoblasts are the principal source of NOTUM in cortical bone, and NOTUM derived from osteocytes/late osteoblasts reduces cortical bone mass. These findings demonstrate that inhibition of osteocyte/late osteoblast-derived NOTUM might be an interesting pharmacological target to increase cortical bone mass and reduce nonvertebral fracture risk.NEW & NOTEWORTHY NOTUM produced by osteoblasts is known to regulate cortical bone mass. Our new findings show that NOTUM specifically derived by DMP1-expressing osteocytes and late osteoblasts regulates cortical bone mass and not trabecular bone mass.

Immunoglobulin G complexes without sialic acids enhance osteoclastogenesis but do not affect arthritis-mediated bone loss.

Immunoglobulin G (IgG) is important in clearance and recognition of previously presented antigens and after activation, IgGs can interact with the Fc gamma receptors (FcγRs) on haematopoietic cells, including bone-resorbing osteoclasts. The pathogenicity of IgG, that is the ability to elicit stimulatory effects via FcγRs, can be modulated by attachment of sugar moieties, including sialic acids. Human IgGs and autoantibodies are associated with bone loss in autoimmune disease. However, the impact of polyclonal murine IgG via FcγRs on bone loss is poorly understood. Here, we investigate if heat-aggregated activated murine polyclonal IgG complexes have any direct effects on murine osteoclasts and if they modulate arthritis-mediated bone loss. Using cell cultures of murine osteoclasts, we show that IgG complexes without sialic acids (de-IgG complexes) enhance receptor activator of nuclear factor kappa-Β ligand (RANKL)-stimulated osteoclastogenesis, an effect associated with increased FcγRIII expression. Using an in vivo model of arthritis-mediated bone loss, where IgG complexes were injected into arthritic knees, no effect on the severity of arthritis or the degree of arthritis-mediated bone loss was detected. Interestingly, injection of de-IgG complexes into non-arthritic knees increased osteoclast formation and enhanced bone erosions. Our findings show that activated de-IgG complexes have no additive effect on arthritis-mediated bone loss. However, de-IgG complexes potentiate murine osteoclastogenesis and enhance local bone erosion in non-arthritic bones, further confirming the link between the adaptive immune system and bone.

Glucocorticoids employ the monomeric glucocorticoid receptor to potentiate vitamin D3 and parathyroid hormone-induced osteoclastogenesis.

Glucocorticoid (GC) therapy decreases bone mass and increases the risk of fractures. We investigated interactions between the GC dexamethasone (DEX) and the bone resorptive agents 1,25(OH)2-vitamin D3 (D3) and parathyroid hormone (PTH) on osteoclastogenesis. We observed a synergistic potentiation of osteoclast progenitor cell differentiation and formation of osteoclasts when DEX was added to either D3- or PTH-treated mouse bone marrow cell (BMC) cultures. Cotreatment of DEX with D3 or PTH increased gene encoding calcitonin receptor (Calcr), acid phosphatase 5, tartrate resistant (Acp5), cathepsin K (Ctsk), and TNF superfamily member 11 (Tnfsf11) mRNA, receptor activator of NF-κB ligand protein (RANKL), numbers of osteoclasts on plastic, and pit formation and release of C-terminal fragment of type I collagen from cells cultured on bone slices. Enhanced RANKL protein expression caused by D3 and DEX was absent in BMC from mice in which the GC receptor (GR) was deleted in stromal cells/osteoblasts. Synergistic interactions between DEX and D3 on RANKL and osteoclast formation were present in BMC from mice with attenuated GR dimerization. These data demonstrate that the GR cooperates with D3 and PTH signaling, causing massive osteoclastogenesis, which may explain the rapid bone loss observed with high dosages of GC treatment.-Conaway, H. H., Henning, P., Lie, A., Tuckermann, J., Lerner, U. H. Glucocorticoids employ the monomeric glucocorticoid receptor to potentiate vitamin D3 and parathyroid hormone-induced osteoclastogenesis.

Vitamin A decreases the anabolic bone response to mechanical loading by suppressing bone formation.

Increased vitamin A consumption is associated with decreased cortical bone mass and increased fracture risk in humans. Rodent studies have demonstrated that hypervitaminosis A increases cortical bone resorption, whereas the importance of the effects on bone formation is less well defined. We used an experimental model of increased bone formation by loading of the tibiae to investigate the effect of vitamin A on bone formation. Control [retinol activity equivalents (RAE) 4.5 µg/g chow] or vitamin A (RAE 60 µg/g chow) diets were given to female C57BL/6N mice for 4 wk, after which the tibiae were subjected to axial loading on alternate days for 2 wk, while the diets were continued. Vitamin A inhibited the loading-induced increase in trabecular and cortical bone volume. This was attributed to inhibition of loading-induced increase in osteoblast number and activity, and expression of osteoblastic genes Sp7, Alpl, and Col1a1 in cortical bone. Vitamin A, loading, and combination thereof also resulted in site-specific effects on bone composition measured by Raman spectroscopy. In summary, a clinically relevant dose of vitamin A suppresses the loading-induced gain of bone mass by decreasing bone formation. These observations may have implications for regulation of bone mass caused by physical activity and the risk of osteoporosis in humans.-Lionikaite, V., Henning, P., Drevinge, C., Shah, F. A., Palmquist, A., Wikström, P., Windahl, S. H., Lerner, U. H. Vitamin A decreases the anabolic bone response to mechanical loading by suppressing bone formation.

Osteoblast-derived NOTUM reduces cortical bone mass in mice and the NOTUM locus is associated with bone mineral density in humans.

Osteoporosis is a common skeletal disease, affecting millions of individuals worldwide. Currently used osteoporosis treatments substantially reduce vertebral fracture risk, whereas nonvertebral fracture risk, mainly caused by reduced cortical bone mass, has only moderately been improved by the osteoporosis drugs used, defining an unmet medical need. Because several wingless-type MMTV integration site family members (WNTs) and modulators of WNT activity are major regulators of bone mass, we hypothesized that NOTUM, a secreted WNT lipase, might modulate bone mass via an inhibition of WNT activity. To characterize the possible role of endogenous NOTUM as a physiologic modulator of bone mass, we developed global, cell-specific, and inducible Notum-inactivated mouse models. Notum expression was high in the cortical bone in mice, and conditional Notum inactivation revealed that osteoblast lineage cells are the principal source of NOTUM in the cortical bone. Osteoblast lineage-specific Notum inactivation increased cortical bone thickness via an increased periosteal circumference. Inducible Notum inactivation in adult mice increased cortical bone thickness as a result of increased periosteal bone formation, and silencing of Notum expression in cultured osteoblasts enhanced osteoblast differentiation. Large-scale human genetic analyses identified genetic variants mapping to the NOTUM locus that are strongly associated with bone mineral density (BMD) as estimated with quantitative ultrasound in the heel. Thus, osteoblast-derived NOTUM is an essential local physiologic regulator of cortical bone mass via effects on periosteal bone formation in adult mice, and genetic variants in the NOTUM locus are associated with BMD variation in adult humans. Therapies targeting osteoblast-derived NOTUM may prevent nonvertebral fractures.-Movérare-Skrtic, S., Nilsson, K. H., Henning, P., Funck-Brentano, T., Nethander, M., Rivadeneira, F., Coletto Nunes, G., Koskela, A., Tuukkanen, J., Tuckermann, J., Perret, C., Souza, P. P. C., Lerner, U. H., Ohlsson, C. Osteoblast-derived NOTUM reduces cortical bone mass in mice and the NOTUM locus is associated with bone mineral density in humans.

The critical interplay between bone resorbing and bone forming cells.

AIM: In this article, the interplay between bone resorbing and bone forming cells is reviewed. METHOD: This review examines the comprehensive literature on the interaction between bone resorption and bone formation. RESULTS: Coupling between bone resorption and bone formation refers to the process within basic multicellular units, in which osteoclastic bone resorption is met by the differentiation of osteoblasts and their bone forming activity. There are many possible signalling molecules that contribute to coupling at the asynchronously working remodelling sites throughout our skeleton. These include growth factors released from the bone matrix during bone resorption, soluble and membrane products of the osteoclasts and their precursors and signals from osteocytes. CONCLUSIONS: In this review, we describe the potential roles of a number of these factors, whose interactions are essential for a tight control of coupling within individual remodelling units, in order to control skeletal mass. Both pre-clinical evidence and clinical evidence pinpoint that molecules in the WNT signalling pathway could be promising bone augmentation therapeutic targets. Regarding oral implications, there is support, from preclinical studies in rats, that anti-sclerostin antibodies can restore alveolar bone mass.

Osteoclasts in Health and Disease.

Osteoclasts are multinucleated, giant cells originating from myeloid hematopoetic stem cells. These are the only cells in nature which can resorb bone. Differentiation of mononucleated osteoclast progenitor cells requires stimulation with M-CSF (macrophage colony-stimulating factor) for the cells to proliferate and survive and with RANKL (receptor activator of nuclear factor kappa B ligand) for differentiation along the osteoclastic lineage to cells which eventually fuse to the mature, multinucleated osteoclasts. Therefore, most hormones and cytokines stimulating osteoclastogenesis do so indirectly by regulating the expression in osteoblasts of RANKL and its inhibitory decoy receptor OPG. Antibodies neutralizing RANKL is a common therapy to inhibit excessive osteoclast formation in diseases such as osteoporosis and malignant tumors with skeletal metastasis. Mature osteoclasts resorb bone by stimulating acid release into the resorption lacunae, followed by proteolytic degradation of bone matrix proteins. Loss-of-function mutations of proteins involved in acidification and proteolysis cause osteopetrosis, a disease with sclerotic bone due to non-functional osteoclasts. Osteoclasts are important for a healthy skeleton by removing damaged bone during remodeling of the skeleton, but are also important for modeling of bone, calcium homeostasis and tooth eruption, and possibly also for glucose and fat metabolism. Loss of bone in inflammatory disease, metastasizing tumors and osteoporosis is due to increased RANKL expression and enhanced osteoclast formation. The present overview aims to summarize how osteoclasts are formed and resorb bone in health and disease.

Antibiotics with Interleukin-15 Inhibition Reduce Joint Inflammation and Bone Erosions but Not Cartilage Destruction in Staphylococcus aureus-Induced Arthritis.

Staphylococcus aureus-induced arthritis causes rapid joint destruction, often leading to disabling joint damage despite antibiotics. We have previously shown that interleukin-15 (IL-15) inhibition without antibiotics is beneficial in S. aureus-induced arthritis. We therefore hypothesized that the inhibition of IL-15, in combination with antibiotics, might represent a useful therapy that would reduce inflammation and joint destruction but preserve the host's ability to clear the infection. Female wild-type C57BL/6 mice were intravenously inoculated with the toxic shock syndrome toxin 1 (TSST-1)-producing LS-1 strain of S. aureus with 0.8 × 108 CFU S. aureus LS-1/mouse. Three days later, treatment consisting of cloxacillin, followed by flucloxacillin, together with either anti-IL-15 antibodies (aIL-15ab) or control antibodies, was started. Studied outcomes included survival, weight change, bacterial clearance, and joint damage. The addition of aIL-15ab to antibiotics in S. aureus-induced arthritis reduced synovitis and bone erosions compared to controls. The number of bone-resorbing osteoclasts in the joints was reduced, whereas cartilage destruction was not significantly altered. Importantly, the combination therapy did not adversely affect the clinical outcome of S. aureus-induced arthritis, such as survival or weight change, or compromise the host's ability to clear the infection. Since the clinical outcome of S. aureus-induced arthritis was not affected, the addition of aIL-15ab to antibiotics ought to be safe. Taken together, the combination of aIL-15ab and antibiotics is a beneficial, but not optimal, treatment of S. aureus-induced arthritis since it reduces synovitis and bone erosions but has a limited effect on cartilage destruction.

WNT16 overexpression partly protects against glucocorticoid-induced bone loss.

Therapeutic use of glucocorticoids (GCs) is a major cause of secondary osteoporosis, but the molecular mechanisms responsible for the deleterious effects of GCs in bone are only partially understood. WNT16 is a crucial physiological regulator of bone mass and fracture susceptibility, and we hypothesize that disturbed WNT16 activity might be involved in the deleterious effects of GC in bone. Twelve-week-old female Obl-Wnt16 mice (WNT16 expression driven by the rat procollagen type I α1 promoter) and wild-type (WT) littermates were treated with prednisolone (7.6 mg·kg-1·day-1) or vehicle for 4 wk. We first observed that GC treatment decreased the Wnt16 mRNA levels in bone of female mice (-56.4 ± 6.1% compared with vehicle, P < 0.001). We next evaluated if WNT16 overexpression protects against GC-induced bone loss. Dual-energy X-ray absorptiometry analyses revealed that GC treatment decreased total body bone mineral density in WT mice (-3.9 ± 1.2%, P = 0.028) but not in Obl-Wnt16 mice (+1.3 ± 1.4%, nonsignificant). Microcomputed tomography analyses showed that GC treatment decreased trabecular bone volume fraction (BV/TV) of the femur in WT mice ( P = 0.019) but not in Obl-Wnt16 mice. Serum levels of the bone formation marker procollagen type I N-terminal propeptide were substantially reduced by GC treatment in WT mice (-50.3 ± 7.0%, P = 0.008) but not in Obl-Wnt16 mice (-3.8 ± 21.2%, nonsignificant). However, the cortical bone thickness in femur was reduced by GC treatment in both WT mice and Obl-Wnt16 mice. In conclusion, GC treatment decreases Wnt16 mRNA levels in bone and WNT16 overexpression partly protects against GC-induced bone loss.

The bone-sparing effects of estrogen and WNT16 are independent of each other.

Wingless-type MMTV integration site family (WNT)16 is a key regulator of bone mass with high expression in cortical bone, and Wnt16(-/-) mice have reduced cortical bone mass. As Wnt16 expression is enhanced by estradiol treatment, we hypothesized that the bone-sparing effect of estrogen in females is WNT16-dependent. This hypothesis was tested in mechanistic studies using two genetically modified mouse models with either constantly high osteoblastic Wnt16 expression or no Wnt16 expression. We developed a mouse model with osteoblast-specific Wnt16 overexpression (Obl-Wnt16). These mice had several-fold elevated Wnt16 expression in both trabecular and cortical bone compared with wild type (WT) mice. Obl-Wnt16 mice displayed increased total body bone mineral density (BMD), surprisingly caused mainly by a substantial increase in trabecular bone mass, resulting in improved bone strength of vertebrae L3. Ovariectomy (ovx) reduced the total body BMD and the trabecular bone mass to the same degree in Obl-Wnt16 mice and WT mice, suggesting that the bone-sparing effect of estrogen is WNT16-independent. However, these bone parameters were similar in ovx Obl-Wnt16 mice and sham operated WT mice. The role of WNT16 for the bone-sparing effect of estrogen was also evaluated in Wnt16(-/-) mice. Treatment with estradiol increased the trabecular and cortical bone mass to a similar extent in both Wnt16(-/-) and WT mice. In conclusion, the bone-sparing effects of estrogen and WNT16 are independent of each other. Furthermore, loss of endogenous WNT16 results specifically in cortical bone loss, whereas overexpression of WNT16 surprisingly increases mainly trabecular bone mass. WNT16-targeted therapies might be useful for treatment of postmenopausal trabecular bone loss.

Bone Cell Activity in Clinical Prostate Cancer Bone Metastasis and Its Inverse Relation to Tumor Cell Androgen Receptor Activity.

Advanced prostate cancer frequently metastasizes to bone and induces a mixed osteoblastic/osteolytic bone response. Standard treatment for metastatic prostate cancer is androgen-deprivation therapy (ADT) that also affects bone biology. Treatment options for patients relapsing after ADT are limited, particularly in cases where castration-resistance does not depend on androgen receptor (AR) activity. Patients with non-AR driven metastases may, however, benefit from therapies targeting the tumor microenvironment. Therefore, the current study specifically investigated bone cell activity in clinical bone metastases in relation to tumor cell AR activity, in order to gain novel insight into biological heterogeneities of possible importance for patient stratification into bone-targeting therapies. Metastasis tissue obtained from treatment-naïve (n = 11) and castration-resistant (n = 28) patients was characterized using whole-genome expression analysis followed by multivariate modeling, functional enrichment analysis, and histological evaluation. Bone cell activity was analyzed by measuring expression levels of predefined marker genes representing osteoclasts (ACP5, CTSK, MMP9), osteoblasts (ALPL, BGLAP, RUNX2) and osteocytes (SOST). Principal component analysis indicated a positive correlation between osteoblast and osteoclast activity and a high variability in bone cell activity between different metastases. Immunohistochemistry verified a positive correlation between runt-related transcription factor 2 (RUNX2) positive osteoblasts and tartrate-resistant acid phosphatase (TRAP, encoded by ACP5) positive osteoclasts lining the metastatic bone surface. No difference in bone cell activity was seen between treatment-naïve and castration-resistant patients. Importantly, bone cell activity was inversely correlated to tumor cell AR activity (measured as AR, FOXA1, HOXB13, KLK2, KLK3, NKX3-1, STEAP2, and TMPRSS2 expression) and to patient serum prostate-specific antigen (PSA) levels. Functional enrichment analysis indicated high bone morphogenetic protein (BMP) signaling in metastases with high bone cell activity and low tumor cell AR activity. This was confirmed by BMP4 immunoreactivity in tumor cells of metastases with ongoing bone formation, as determined by histological evaluation of van Gieson-stained sections. In conclusion, the inverse relation observed between bone cell activity and tumor cell AR activity in prostate cancer bone metastasis may be of importance for patient response to AR and/or bone targeting therapies, but needs to be evaluated in clinical settings in relation to serum markers for bone remodeling, radiography and patient response to therapy. The importance of BMP signaling in the development of sclerotic metastasis lesions deserves further exploration.

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.

TLR5, a novel mediator of innate immunity-induced osteoclastogenesis and bone loss.

Accumulating evidence points to the importance of the innate immune system in inflammation-induced bone loss in infectious and autoimmune diseases. TLRs are well known for being activated by ligands expressed by bacteria, viruses, and fungi. Recent findings indicate that also endogenous ligands in inflammatory processes are important, one being a TLR5 agonist present in synovial fluid from patients with rheumatoid arthritis (RA). We found that activation of TLR5 by its specific ligand, flagellin, caused robust osteoclast formation and bone loss in cultured mouse neonatal parietal bones dependent on increased receptor activator of NF-κB ligand (RANKL):osteoprotegerin ratio, with half-maximal stimulation at 0.01 µg/ml. Flagellin enhanced Rankl mRNA in isolated osteoblasts by a myeloid differentiation primary response gene 88 and NF-κB-dependent mechanism. Injection of flagellin locally over skull bones in 5-wk-old mice resulted in increased mRNA expression of Rankl and osteoclastic genes, robust osteoclast formation, and bone loss. The effects in vitro and in vivo were absent in Tlr5(-/-) mice. These data show that TLR5 is a novel activator of RANKL and osteoclast formation and, therefore, a potential key factor in inflammation-induced bone erosions in diseases like RA, reactive arthritis, and periodontitis. TLR5 might be a promising novel treatment target for prevention of inflammatory bone loss.

Osteoblast differentiation is enhanced by a nano-to-micro hybrid titanium surface created by Yb:YAG laser irradiation.

OBJECTIVES: The aim of this study was to analyze the capacity of a new modified laser surface to stimulate calvarial osteoblasts isolated from neonatal mouse bones to differentiate and form mineralized nodules. METHODS: Titanium discs were subjectezd or not to laser irradiation according to specific parameters and characterized. Osteoblasts isolated from neonatal mouse calvaria were cultured over the discs, and the capacity of these cells to proliferate (MTT assay), form mineralized nodules (Alizarin red assay), and enhance alkaline phosphatase activity (ALPase activity) was analyzed. Real-time PCR was used for quantification of gene expression. RESULTS: Laser-irradiated titanium discs (L) presented a rough nano-to-micrometric oxidized surface contrasting with the smooth pattern on polished discs (P). The Ra on the micrometric level increased from 0.32 ± 0.01 µm on P surfaces to 10.57 ± 0.39 µm on L surfaces. When compared with P, L promoted changes in osteoblast morphology, increased mineralized nodule formation in osteoblasts cultured on the surfaces for 14 days, and enhanced ALPase activity at days 7 and 14. Transcription factors triggering osteoblast differentiation (Runx2 and Sp7) and genes encoding the bone extracellular matrix proteins collagen type-1 (Col1a1), osteopontin (Spp1), and osteocalcin (Bglap) were upregulated in cells on L surfaces compared with those on P surfaces at days 1-14. CONCLUSION: Laser treatment of titanium surfaces created a rough surface that stimulated osteoblast differentiation. CLINICAL RELEVANCE: Laser treatment of titanium generates a reproducible and efficient surface triggering osteoblast differentiation that can be of importance for osteointegration.

Cysteine proteinase inhibitors regulate human and mouse osteoclastogenesis by interfering with RANK signaling.

The cysteine proteinase inhibitor cystatin C inhibited RANKL-stimulated osteoclast formation in mouse bone marrow macrophage cultures, an effect associated with decreased mRNA expression of Acp5, Calcr, Ctsk, Mmp9, Itgb3, and Atp6i, without effect on proliferation or apoptosis. The effects were concentration dependent with half-maximal inhibition at 0.3 µM. Cystatin C also inhibited osteoclast formation when RANKL-stimulated osteoclasts were cultured on bone, leading to decreased formation of resorption pits. RANKL-stimulated cells retained characteristics of phagocytotic macrophages when cotreated with cystatin C. Three other cysteine proteinase inhibitors, cystatin D, Z-RLVG-CHN2 (IC50 0.1 µM), and E-64 (IC50 3 µM), also inhibited osteoclast formation in RANKL-stimulated macrophages. In addition, cystatin C, Z-RLVG-CHN2, and E-64 inhibited osteoclastic differentiation of RANKL-stimulated CD14(+) human monocytes. The effect by cystatin C on differentiation of bone marrow macrophages was exerted at an early stage after RANKL stimulation and was associated with early (4 h) inhibition of c-Fos expression and decreased protein and nuclear translocation of c-Fos. Subsequently, p52, p65, IκBα, and Nfatc1 mRNA were decreased. Cystatin C was internalized in osteoclast progenitors, a process requiring RANKL stimulation. These data show that cystatin C inhibits osteoclast differentiation and formation by interfering intracellularly with signaling pathways downstream RANK.