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In this review we aim to summarize the latest findings on the network of molecules produced by muscle and bone under physiological and pathological conditions. The concomitant onset of osteoporosis and sarcopenia is currently one of the main threats that can increase the risk of falling fractures during aging, generating high health care costs due to hospitalization for bone fracture surgery. With the growing emergence of developing innovative therapies to treat these two age-related conditions that often have common onset, a broader understanding of molecular messengers regulating the communication between muscle and bone tissue became imperative. Recently it has been highlighted that two muscle-derived signals, such as the myokines Irisin and L-BAIBA, positively affect bone tissue. In parallel, there are signals derived from bone that affect either positively the skeletal muscle, such as osteocalcin, or negatively, such as RANKL.

Bone loss induced by ovariectomy is due to the direct activity on bone cells and mesenchymal cells and to the dysregulated activity of bone marrow cells, including immune cells and stromal cells, but the underlying mechanisms are not completely known. Here, we demonstrate that ovariectomy induces the T-cell co-stimulatory cytokine LIGHT, which stimulates both osteoblastogenesis and osteoclastogenesis by modulating osteoclastogenic cytokine expression, including TNF, osteoprotegerin, and the receptor activator of nuclear factor-κB ligand (RANKL). Predictably, LIGHT-deficient (Tnfsf14-/- ) mice are protected from ovariectomy-dependent bone loss, whereas trabecular bone mass increases in mice deficient in both LIGHT and T and B lymphocytes (Rag -/- Tnfsf14 -/- ) and is associated with an inversion of the TNF and RANKL/OPG ratio. Furthermore, women with postmenopausal osteoporosis display high levels of LIGHT in circulating T cells and monocytes. Taken together, these results indicate that LIGHT mediates bone loss induced by ovariectomy, suggesting that patients with postmenopausal osteoporosis may benefit from LIGHT antagonism. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tumor necrosis factor superfamily member 14 (TNFSF14), LIGHT, is a component of the cytokine network that regulates innate and adaptive immune responses, which promote homeostasis of lymphoid organs, liver, and bone. Metastatic tumors often disrupt the tissue microenvironment, thus altering the homeostasis of the invaded organ; however, the underlying mechanisms required further studies. We investigated the role of LIGHT in osteolytic bone disease induced by metastatic non-small cell lung cancer (NSCLC). Patients diagnosed with NSCLC bone metastasis show significantly higher levels of LIGHT expressed in monocytes compared with non-bone metastatic tumors and healthy controls. Serum LIGHT levels were also higher in patients with bone metastases than in controls, suggesting a role for LIGHT in stimulating osteoclast precursors. In bone metastatic patients, we also detected increased RNA expression and serum RANKL levels, thus by adding anti-LIGHT or RANK-fragment crystallizable region (RANK-Fc) in PBMC cultures, a significant inhibition of osteoclastogenesis was observed. To model this observation in mice, we used the mouse lung cancer cell line LLC-1. After intratibial implantation, wild-type mice showed an increased number of osteoclasts but reduced numbers of osteoblasts and decreased osteoid formation. In contrast, Tnfsf14-/- mice showed no significant bone loss or other changes in bone homeostasis associated with this model. These data indicate LIGHT is a key control mechanism for regulating bone homeostasis during metastatic invasion. Thus, LIGHT may be a novel therapeutic target in osteolytic bone metastases. © 2019 American Society for Bone and Mineral Research.

To date, pharmacological strategies designed to accelerate bone fracture healing are lacking. We subjected 8-week-old C57BL/6 male mice to closed, transverse, mid-diaphyseal tibial fractures and treated them with intraperitoneal injection of a vehicle or r-irisin (100 µg/kg/weekly) immediately following fracture for 10 days or 28 days. Histological analysis of the cartilaginous callus at 10 days showed a threefold increase in Collagen Type X (p = 0.0012) and a reduced content of proteoglycans (40%, p = 0.0018). Osteoclast count within the callus showed a 2.4-fold increase compared with untreated mice (p = 0.026), indicating a more advanced stage of endochondral ossification of the callus during the early stage of fracture repair. Further evidence that irisin induced the transition of cartilage callus into bony callus was provided by a twofold reduction in the expression of SOX9 (p = 0.0058) and a 2.2-fold increase in RUNX2 (p = 0.0137). Twenty-eight days post-fracture, microCT analyses showed that total callus volume and bone volume were increased by 68% (p = 0.0003) and 67% (p = 0.0093), respectively, and bone mineral content was 74% higher (p = 0.0012) in irisin-treated mice than in controls. Our findings suggest that irisin promotes bone formation in the bony callus and accelerates the fracture repair process, suggesting a possible use as a novel pharmacologic modulator of fracture healing.

Simple Summary Irisin is a recently discovered protein, mainly produced in the muscle tissue, whose action is proving effective in many other tissues. The crosstalk between muscle and bone has been long since demonstrated, and physical activity has shown to have an impressive positive effect in both tissues. Irisin production increases with exercising and drops with sedentariness and aging, indicating that the molecule is involved in sarcopenia and in bone mass reduction. Although skeleton is target of irisin, its mechanism of action on bone cells has not yet been completely elucidated. The aim of this work is to analyze the effect of irisin on osteoblast differentiation; to this purpose, we used a stem cell model reproducing the osteoblastogenesis and the bone-forming processes. We performed an in vitro study exploring the main osteoblast markers in the presence of irisin. We found that irisin has an impressive effect on the most peculiar osteoblast feature: the bone mineral matrix secretion process. Moreover, irisin demonstrated an inductive effect on osteoblast osteocalcin production. Both results suggest a stimulating effect of irisin in bone formation. The association we observed between irisin addition and osteoblast osteocalcin production should be further investigated. Abstract The myokine irisin, well known for its anabolic effect on bone tissue, has been demonstrated to positively act on osteoblastic differentiation processes in vitro. Mesenchymal stem cells (MSCs) have captured great attention in precision medicine and translational research for several decades due to their differentiation capacity, potent immunomodulatory properties, and their ability to be easily cultured and manipulated. Dental bud stem cells (DBSCs) are MSCs, isolated from dental tissues, that can effectively undergo osteoblastic differentiation. In this study, we analyzed, for the first time, the effects of irisin on DBSC osteogenic differentiation in vitro. Our results indicated that DBSCs were responsive to irisin, showed an enhanced expression of osteocalcin (OCN), a late marker of osteoblast differentiation, and displayed a greater mineral matrix deposition. These findings lead to deepening the mechanism of action of this promising molecule, as part of osteoblastogenesis process. Considering the in vivo studies of the effects of irisin on skeleton, irisin could improve bone tissue metabolism in MSC regenerative procedures.

Irisin, the circulating peptide originating from fibronectin type III domain-containing protein 5 (FNDC5), is mainly expressed by muscle fibers under peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) control during exercise. In addition to several beneficial effects on health, physical activity positively affects nervous system functioning, particularly the hippocampus, resulting in amelioration of cognition impairments. Recently, FNDC5/irisin detection in hippocampal neurons and the presence of irisin in the cerebrospinal fluid opened a new intriguing chapter in irisin history. Interestingly, in the hippocampus of mice, exercise increases FNDC5 levels and upregulates brain-derived neurotrophic factor (BDNF) expression. BDNF, displaying neuroprotection and anti-inflammatory effects, is mainly produced by microglia and astrocytes. In this review, we discuss how these glial cells can morphologically and functionally switch during neuroinflammation by modulating the expression of a plethora of neuroprotective or neurotoxic factors. We also focus on studies investigating the irisin role in neurodegenerative diseases (ND). The emerging involvement of irisin as a mediator of the multiple positive effects of exercise on the brain needs further studies to better deepen this issue and the potential use in therapeutic approaches for neuroinflammation and ND.

Understanding molecular mechanisms responsible for bone cells unbalance in microgravity would allow the development of better countermeasures for astronauts, and eventually advancing terrestrial osteoporosis treatments. We conduct a unique investigation by using a controlled 3D in vitro cell model to mimic the bone microenvironment in microgravity aboard the SpaceX Dragon cargo ferry to the ISS. Osteoblasts (OBs), osteoclasts (OCs), and endothelial cells (ECs), seeded on Skelite discs, were cultured w/ or w/o rec-Irisin and exposed to 14 days of microgravity in the eOSTEO hardware. Gene expression analysis was assessed, and results were compared to ground controls treated within identical payloads. Our results show that the microgravity-induced downregulation of mRNA levels of genes encoding for OB key transcription factors (Atf4 -75%, P < .01; RunX2 -87%, P < .001, Osterix -95%, P < .05 vs ground) and proteins (Collagen I -84%, P < .05; Osteoprotegerin -94%, P < .05) were prevented by irisin. Despite it was not effective in preventing Trap and Cathepsin K mRNA increase, irisin induced a 2.8-fold increase of Osteoprotegerin (P < .05) that might act for reducing osteoclastogenesis in microgravity. Our results provide evidence that irisin supports OB differentiation and activity in microgravity and it might represent a countermeasure to prevent bone loss in astronauts.

The human skeleton, consisting of several hundreds of bones, is a high dynamic system absolving to different functions, such as locomotion, reservoir of minerals, hematopoiesis, etc. Skeleton shows a sexual dimorphic phenotype: It is larger and more robust in men compared to women. In humans, differences in the skeleton size are well represented by stature, but the most important differences are in the bone geometry and microarchitecture, determined by the peak bone mass achievement in young adulthood and by the subsequent turnover in cortical and trabecular bones, which result in the substantial gender difference in bone strength. Estrogens and androgens affect skeleton growth and maintenance and are responsible for the sexual dimorphism.

Peroxisome proliferator-activated receptor-gamma coactivator (PGC1α) is a transcription coactivator that interacts with a broad range of transcription factors involved in several biological responses. Here, we show that PGC1α plays a role in skeletal homeostasis since aged PGC1α-deficient mice (PGC1α−/−) display impaired bone structure. Micro-CT of the tibial mid-shaft showed a marked decrease of cortical thickness in PGC1α−/− (− 11.9%, p

Alkaptonuria (AKU) is a rare disorder characterized by the deficiency of the enzyme homogentisate 1,2-dioxygenase and consequent homogentisate accumulation, which leads to progressive and severe osteoarthopathy starting from the second decade of life. Thus, in AKU patients, bone involvement represents an important clinical issue, which we investigated. Serum levels of receptor activator of NF-κB ligand (RANKL), osteoprotegerin, sclerostin, Dickkopf-1, and bone remodeling markers were measured in nine AKU patients (two children and seven adults) and 22 controls, together with lumbar spine bone mineral density (LS-BMD) and femoral-BMD. In the two AKU children, the average of LS-BMD and femoral-BMD Z-scores were within the normal range, but reduced with respect to the controls. Otherwise, in the adult AKU patients, LS-BMD T-score was inside the normal range, but femoral-BMD T-score reached osteopenic levels. Consistently, in AKU adults, higher RANKL and C-terminal telopeptide of collagen type 1 and lower osteoprotegerin levels were observed than in controls. Otherwise, spontaneous osteoclastogenesis was already evident in peripheral blood mononuclear cell cultures from AKU children, together with a high percentage of circulating osteoclast precursors. Osteoclastogenesis was sustained by the high levels of tumor necrosis factor-α, RANK, RANKL, and LIGHT. In conclusion, the altered osteoclastogenesis was observed already in AKU children, despite the absence of evident injury. Thus, a preventive approach in young patients, targeting osteoclast activity, may prevent the macroscopic bone disease that appears in adult AKU.

Irisin is a myokine secreted by the skeletal muscle during physical activity both in mice and humans. Its first identified role was to activate the browning response in white adipocytes, subsequently triggering non-shivering thermogenesis; therefore, Irisin has raised great expectations as a potential target in the treatment of obesity. In 2015, we demonstrated that Irisin plays a central role in the control of bone mass, driving positive effects on cortical mineral density and bone mechanical properties. This effect on the bone was triggered using an Irisin dosage 70 times lower than the one needed to induce the browning response, suggesting that the skeleton is the primary target organ of this myokine. Moreover, our studies also highlighted the autocrine effect of Irisin on the skeletal muscle, overall suggesting that Irisin plays a fundamental role in the physiology of the musculoskeletal system. More recently, we demonstrated the efficacy of Irisin in preventing and restoring bone and muscle losses in a mouse model affected by disuse-induced osteoporosis and muscular atrophy. Hopefully, if future investigations will be confirmed in humans, it may lead to develop an Irisin-based therapy for physically disable or bedridden patients and it might also represent a countermeasure for astronauts subjected to microgravity.

Multiple cytokines produced by immune cells induce remodeling and aid in maintaining bone homeostasis through differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. Here, we investigate bone remodeling controlled by the tumor necrosis factor (TNF) superfamily cytokine LIGHT. LIGHT-deficient mice (Tnfsf14-/- ) exhibit spine deformity and reduced femoral cancellous bone mass associated with an increase in the osteoclast number and a slight decrease of osteoblasts compared with WT mice. The effect of LIGHT in bone cells can be direct or indirect, mediated by both the low expression of the anti-osteoclastogenic osteoprotegerin (OPG) in B and T cells and reduced levels of the pro-osteoblastogenic Wnt10b in CD8+ T cells in Tnfsf14-/- mice. LIGHT stimulation increases OPG levels in B, CD8+ T, and osteoblastic cells, as well as Wnt10b expression in CD8+ T cells. The high bone mass in Light and T- and B-cell-deficient mice (Rag- /Tnfsf14- ) supports the cooperative role of the immune system in bone homeostasis. These results implicate LIGHT as a potential target in bone disease. © 2017 American Society for Bone and Mineral Research.

Multiple myeloma (MM) is a hematologic malignancy of plasma cells proliferating in the bone marrow and it is associated with the osteolytic bone disease. About 70% of myeloma patients show the bone disease at diagnosis and 15–20% have pathologic fractures. The osteolytic disease is due to the altered bone remodeling with increased activity of osteoclasts, the bone resorbing cells, and the reduced activity of osteoblasts, the bone forming cells. In the bone marrow microenvironment, the deregulated activity of bone cells is due to the malignant plasma cells through the release of soluble molecules or cell-cell contact. Recently, the key role of osteocytes, the orchestrators of bone remodeling, and the adipocytes has been highlighted in MM-bone disease. In this review, we overview the mechanisms leading to increased bone resorption as well as suppressed osteoblast formation and activity in MM-bone disease, thus highlighting cytokines that will represent new therapeutic targets to improve bone health in MM-bone disease.

Irisin is a hormone-like myokine produced in abundance by skeletal muscle in response to exercise, both in mice and humans. Once released into the circulation, irisin acts on white adipocytes to induce the browning response and subsequently activates nonshivering thermogenesis. We have examined the premise that irisin produced during exercise may subserve further functions in the musculoskeletal system. We review evidence for its possible skeletal effects, including the central role that irisin plays in the control of bone mass, with positive effects on cortical mineral density and geometry in mice. We also review the autocrine effects of irisin in skeletal muscle, in which it upregulates the expression of its precursor (FNDC5). Since loss of bone and muscle mass occurs with aging, immobility, and several metabolic diseases, future studies exploring the efficacy of irisin in restoring bone and reversing muscle wasting could be important to establishing irisin as a molecule that combines beneficial effects for treating osteoporosis and muscular atrophy. If the results from mice were confirmed in human studies, an irisin-based therapy could be developed for physically disabled or bedridden patients.

Previous results showed that intermittently administered irisin improves bone mass in normal mice and prevents the development of disuse-induced osteoporosis and muscular atrophy in hindlimb-suspended mice, a murine model able to mimic the absence of mechanical loading. A recent study showed that irisin increases survival of osteocytes acting through integrin αV/β5 receptors. To better understand the action of irisin on these cells, we investigated the downstream signaling cascades in osteocyte-like cells (MLO-Y4) treated with recombinant irisin (rec-irisin) in vitro and we analyzed survival of osteocytes and caspase activation in cortical bone of osteoporotic mice treated with rec-irisin in vivo. Our results revealed that rec-irisin activated the MAP kinases Erk1 and Erk2 and increased the expression of the transcription factor Atf4 (2.5-fold, p < .05) through an Erk-dependent pathway in osteocytes. Some key genes expressed by MLO-Y4 cells were modulated by long-term irisin treatment, either continuously administered or given with intermittent short pulses. Interestingly, Sost mRNA was severely downregulated only upon intermittent irisin administration (10-fold, p < .001). Furthermore, rec-irisin upregulated Tfam mRNA (fourfold, p < .05) and Bcl2/Bax ratio (twofold, p < .05) in MLO-Y4 cells. By detecting caspase-9 and caspase-3, we also found that rec-irisin inhibited apoptosis induced by hydrogen peroxide and dexamethasone, respectively. In cortical bone of unloading C57BL6 mice treated with vehicle (unload-veh), irisin prevented disuse-induced reduction of viable osteocytes (+30% versus unload-veh, p < .05) and increase of empty lacunae (+110% versus unload-veh, p < .05), as well as caspase-9 (threefold, p < .05) and caspase-3 (twofold, p < .05) activations. Our findings revealed underlying mechanisms of irisin action on osteocytes, which increases their functions and exerts anti-apoptotic effects, confirming that mechanosensor cells of bone are sensitive to the exercise-mimetic myokine irisin. © 2019 American Society for Bone and Mineral Research.

Childhood obesity is one of the major health problems in western countries. The excessive accumulation of adipose tissue causes inflammation, oxidative stress, apoptosis, and mitochondrial dysfunctions. Thus, obesity leads to the development of severe co-morbidities including type 2 diabetes mellitus, liver steatosis, cardiovascular, and neurodegenerative diseases which can develop early in life. Furthermore, obese children have low bone mineral density and a greater risk of osteoporosis and fractures. The knowledge about the interplay bone tissue and between adipose is still growing, although recent findings suggest that adipose tissue activity on bone can be fat-depot specific. Obesity is associated to a low-grade inflammation that alters the expression of adiponectin, leptin, IL-6, Monocyte Chemotactic Protein 1 (MCP1), TRAIL, LIGHT/TNFSF14, OPG, and TNFα. These molecules can affect bone metabolism, thus resulting in osteoporosis. The purpose of this review was to deepen the cellular mechanisms by which obesity may facilitate osteoporosis and bone fractures.

Several lines of evidence have recently established that skeletal muscle is an endocrine organ producing and releasing myokines acting in a paracrine or endocrine fashion. Among these, the newly identified myokine Irisin, produced by skeletal muscle after physical exercise, was originally described as molecule able to promote energy expenditure in white adipose tissue. Recently, it has been shown that the myokine Irisin affects skeletal metabolism in vivo. Thus, mice treated with a micro-dose of r-Irisin displayed improved cortical bone mass, geometry and strength, resembling the effect of physical activity in developing an efficient load-bearing skeleton. Further studies highlighted the autocrine effect of Irisin on skeletal muscle, and research performed in humans has definitively established that Irisin is a circulating hormone-like myokine, increased by physical activity. Albeit there are still few, since Irisin has been very recently discovered, herein are summarized the most relevant research findings published on this topic.

In this study, we investigated the bone cell activity in patients with osteogenesis imperfecta (OI) treated and untreated with neridronate. We demonstrated the key role of Dickkopf-1 (DKK1), receptor activator of nuclear factor-κB ligand (RANKL), and tumor necrosis factor alpha (TNF-α) in regulating bone cell of untreated and treated OI subjects. These cytokines could represent new pharmacological targets for OI. Bisphosphonates are widely used in the treatment of children with osteogenesis imperfecta (OI) with the objective of reducing the risk of fractures. Although bisphosphonates increase bone mineral density in OI subjects, the effects on fracture incidence are conflicting. The aim of this study was to investigate the mechanisms underlying bone cell activity in subjects with mild untreated forms of OI and in a group of subjects with severe OI treated with cycles of intravenous neridronate. Sclerostin, DKK1, TNF-α, RANKL, osteoprotegerin (OPG), and bone turnover markers were quantified in serum of 18 OI patients (12 females, mean age 8.86 ± 3.90), 8 of which were receiving cyclic intravenous neridronate, and 21 sex- and age-matched controls. The effects on osteoblastogenesis and OPG expression of media conditioned by the serum of OI patients and anti-DKK1 neutralizing antibody were evaluated. Osteoclastogenesis was assessed in cultures from patients and controls. DKK1 and RANKL levels were significantly increased both in untreated and in treated OI subjects with respect to controls. The serum from patients with high DKK1 levels inhibited both osteoblast differentiation and OPG expression in vitro. High RANKL and low OPG messenger RNA (mRNA) levels were found in lymphomonocytes from patients. High amounts of TNF-α were expressed by monocytes, and an elevated percentage of circulating CD11b-CD51/CD61+ osteoclast precursors was observed in patients. Our study demonstrated the key role of DKK1, RANKL, and TNF-α in regulating bone cell activity of subjects with OI untreated and treated with bisphosphonates. These cytokines could represent new pharmacological targets for OI patients.

We have previously shown that through the production of high LIGHT levels, immune cells contribute to both osteoclastogenesis and bone destruction in Multiple Myeloma (MM)-related bone disease. With the aim of further exploring the mechanisms underlying the development of MM-related bone disease, here we focused on a possible role of LIGHT in MM patients with active bone disease despite the treatment received. We detected LIGHT over-expression by circulating CD14+ monocytes from MM patients still showing active bone disease, despite the treatment. In addition, we found over-expression of receptor activator of nuclear factor kappa-B ligand (RANKL), whose pro-osteoclastogenic role is well-known, in T-lymphocytes isolated from the same patients. Although the percentages of circulating osteoclast progenitors, CD14+CD16+ monocytes, were higher in all the MM patients than in the controls spontaneous osteoclastogenesis occurred only in the cultures derived from PBMCs of MM patients with unresponsive bone disease. Of note, in the same cultures osteoclastogenesis was partially or completely inhibited, in a dose-dependent manner, by the addition of RANK-Fc or anti-LIGHT neutralizing antibody, demonstrating the contribution of both LIGHT and RANKL to the enhanced osteoclast formation observed. In addition, high serum levels of TRAP5b and CTX, the two markers of osteoclast activity, were detected in MM patients with bone disease not responsive to treatment. In conclusion, our study indicates a prominent role of LIGHT in the crosstalk among osteoclasts and immune cells, co-involved together with RANKL in the pathophysiological mechanisms leading to MM-related bone disease. This TNF superfamily member may thus be a possible new therapeutic target in MM-related bone disease.

The liver stores iron and senses systemic and tissue iron availability. Hepatocytes control iron homeostasis by producing the peptide hormone hepcidin that controls dietary iron absorption and iron release from intracellular stores. Recent data challenged the exclusive role of hepatocytes in controlling iron levels. Indeed, liver sinusoidal endothelial cells (LSECs) increase BMP2 and BMP6 levels in response to iron, which control hepcidin expression in a paracrine manner. However the molecular mechanism(s) of how BMPs respond to iron levels remain unknown. We established primary murine LSEC cultures and exposed these to iron sources. Unexpectedly, BMP2 mRNA expression is strongly reduced by iron treatment, while BMP6 levels are only mildly increased. This finding suggests that intracellular iron content cannot directly activate BMP2 transcription and only slightly contribute to BMP6 upregulation in LSEC cultures. However, if LSECs are co-cultured with iron-loaded primary hepatocytes the expression of BMP2 and BMP6 is increased and the fold induction of BMP6 is greater compared to LSECs cultured alone, suggesting that the iron status of hepatocytes instructs the LSEC BMP response. These data are supported by findings in a genetic mouse model of iron overload (Slc40a1C326S/C326S). Hepatocytes isolated from Slc40a1C326S/C326S mice display an iron-loaded molecular signature and the expected low mRNA expression of Transferrin Receptor 1 (Tfr1). By contrast, LSECs show high expression of Tfr1, indicating intracellular iron deficiency. Despite this, hepatic BMP levels are increased, suggesting that BMP2 and BMP6 expression are directly related to the intracellular iron content of hepatocytes but not LSECs. RNA-sequencing of isolated hepatic cell populations is ongoing to identify putative hepatocyte regulators involved in the iron-mediated BMP2 and BMP6 regulation. Disclosures Muckenthaler: Silence Therapeutics: Consultancy; Novartis: Research Funding.

Cells from dental tissues have a mesenchymal stem cell (MSC) phenotype, are multipotent and can differentiate into osteoblastic cells, as we have previously found. MSCs, due to their tumor‑homing ability, are currently being used as cell‑based delivery systems for cancer protein therapeutics, such as the TNF‑related apoptosis‑inducing ligand (TRAIL). In the present study we revealed that dental pulp stem cells (DPSCs) expressed TRAIL to a greater extent when they were differentiated into the osteoblastic lineage. TRAIL affected the viability of undifferentiated DPSCs, while osteoblastic differentiated DPSCs were not sensitive to TRAIL. The expression trend of TRAIL receptors underwent changes during the osteoblastic differentiation of DPSCs exhibiting low DcR2 and high DR5 levels in the undifferentiated DPSCs and an opposite scenario was presented in the differentiated cells. The sensitivity of the undifferentiated DPSCs to the TRAIL‑apoptotic effect was also associated with low levels of intracellular anti‑apoptotic proteins, such as c‑FLIP, XIAP and the activation of caspase‑8 and ‑3. DPSC‑differentiated osteoblasts expressing high TRAIL levels were capable to affect the cell viability of the human myeloma cell line H929, thus representing an effective anticancer therapeutic method.

Irisin is a new peptide produced mainly by the skeletal muscle playing an important role both in glucose/energy homeostasis and bone metabolism. Childhood type 1 diabetes mellitus (T1DM) is associated with decreased bone mass. We aimed to evaluate irisin levels in TD1M children and their correlation with bone metabolism and glycaemic control.Ninety-six T1DM subjects (12.2 ± 4 years), 56 on multiple daily injections (MDI), 40 on continuous subcutaneous insulin infusion (CSII), and 34 controls were included in the study. Irisin and bone remodeling markers were quantified in sera from patients and controls. Bone mineral density (BMD) was evaluated by QUS.Increased irisin levels were found in T1DM patients respect to controls (p 0.001). With adjustment for age, irisin levels significantly correlated negatively with HbA1c% (r = -0.105, p 0.001), years of diabetes (r = -0.07, p 0.04), 25(OH)-Vitamin D (r = -0.175, p 0.0001), and positively with BTT-Z-score (r = 0.088, p = 0.016), and osteocalcin (r = 0.059, p 0.04). We detected the highest levels of irisin in CSII patients compared to MDI and controls (p 0.001 and p 0.007 respectively).We demonstrated high irisin levels in T1DM children and the association of highest irisin amounts to a better glycaemic control and bone health in TDM1 subjects on CSII.

The human skeleton, consisting of several hundreds of bones, is no longer believed as a static structural system protecting all soft tissues, but it is now thought as a highly dynamic system. In addition to its roles in locomotion, storage of minerals, and production of blood cells, the skeleton has recently been considered as a new endocrine organ, not only able in its regulation but also involved in the regulation of other organs, and, importantly, it plays a crucial role in the cross talk with the immune system and the skeletal muscle. Bone tissue is a metabolically active organ undergoing continuous remodeling through the balanced activity of osteoclasts, the bone-resorbing cells, and osteoblasts, the bone-forming cells, both orchestrated by the osteocytes, the most abundant cells embedded into the bone matrix. Here, we will provide an overview of the cytokines involved in the recent mechanisms highlighting bone cell differentiation and function.

Osteoporosis is the most widespread skeletal disease requiring innovative therapeutic strategies for its management. The understanding of receptor activator of nuclear factor kappa-B ligand (RANKL) and sclerostin's role in bone cell biology is completely changing the therapeutic landscape. RANKL supports osteoclast formation and activity and is mainly produced by cells of osteoblastic lineage. Sclerostin, an antagonist of the Wnt pathway, has a key role in bone formation and is mainly secreted by osteocytes. High levels of RANKL and sclerostin have been detected in osteoporosis, leading to the production of antibodies able to neutralize their activity.In this review, the authors give an overview and discuss the literature and data on denosumab and romosozumab to treat osteoporosis. Clinical studies indicate that long-term treatment with denosumab causes a continuous increase in bone mineral density with low incidence of adverse effects. Romosozumab treatment gives increases bone formation and improves bone mineral density (BMD) though further studies are needed to better evaluate the adverse effects.Denosumab and romosozumab show promise in the treatment of osteoporosis. Furthermore, their different mechanisms of action compared to existing anti-osteoporotic drugs may permit alternative strategies for osteoporosis treatment down the line

We previously showed that Irisin, a myokine released from skeletal muscle after physical exercise, plays a central role in the control of bone mass. Here we report that treatment with recombinant Irisin prevented bone loss in hind-limb suspended mice when administered during suspension (preventive protocol) and induced recovery of bone mass when mice were injected after bone loss due to a suspension period of 4 weeks (curative protocol). MicroCT analysis of femurs showed that r-Irisin preserved both cortical and trabecular bone mineral density, and prevented a dramatic decrease of the trabecular bone volume fraction. Moreover, r-Irisin protected against muscle mass decline in the hind-limb suspended mice, and maintained the fiber cross-sectional area. Notably, the decrease of myosin type II expression in unloaded mice was completely prevented by r-Irisin administration. Our data reveal for the first time that Irisin retrieves disuse‐induced bone loss and muscle atrophy. These findings may lead to development of an Irisin-based therapy for elderly immobile osteoporotic and physically disable patients, and might represent a countermeasure for astronauts subjected to microgravity-induced bone and muscle losses.

It has been recently demonstrated that exercise activity increases the expression of the myokine Irisin in skeletal muscle, which is able to drive the transition of white to brown adipocytes, likely following a phenomenon of transdifferentiation. This new evidence supports the idea that muscle can be considered an endocrine organ, given its ability to target adipose tissue by promoting energy expenditure. In accordance with these new findings, we hypothesized that Irisin is directly involved in bone metabolism, demonstrating its ability to increase the differentiation of bone marrow stromal cells into mature osteoblasts. Firstly, we confirmed that myoblasts from mice subjected to 3 weeks of free wheel running increased Irisin expression compared to nonexercised state. The conditioned media (CM) collected from myoblasts of exercised mice induced osteoblast differentiationin vitroto a greater extent than those of mice housed in resting conditions. Furthermore, the differentiated osteoblasts increased alkaline phosphatase and collagen I expression by an Irisin-dependent mechanism. Our results show, for the first time, that Irisin directly targets osteoblasts, enhancing their differentiation. This finding advances notable perspectives in future studies which could satisfy the ongoing research of exercise-mimetic therapies with anabolic action on the skeleton.

Background/Objectives Calcific aortic valvular disease (CAVD) is an actively regulated process characterized by the activation of specific osteogenic signaling pathways and apoptosis. We evaluated the involvement in CAVD of the TNF-related apoptosis-inducing ligand (TRAIL), an apoptotic molecule which induces apoptosis by interacting with the death receptor (DR)-4 and DR5, and whose activity is modulated by the decoy receptor (DcR)-1 and DcR2. Methods Sections of calcific and normal aortic valves, obtained at surgery time, were subjected to immunohistochemistry and confocal microscopy for TRAIL immunostaining. Valvular interstitial cells (VICs) isolated from calcific (C-VICs) and normal (N-VICs) aortic valves were investigated for the gene and protein expression of TRAIL receptors. Cell viability was assayed by MTT. Von Kossa staining was performed to verify C-VIC ability to produce mineralized nodules. TRAIL serum levels were detected by ELISA. Results Higher levels of TRAIL were detected in calcific aortic valves and in sera from the same patients respect to controls. C-VICs express significantly higher mRNA and protein levels of DR4, DR5, DcR1, DcR2 and Runx2 compared to N-VICs. C-VICs and N-VICs, cultured in osteogenic medium, express significantly higher mRNA levels of DR4, Runx2 and Osteocalcin compared to baseline. C-VICs and N-VICs were sensitive to TRAIL-apoptotic effect at baseline and after osteogenic differentiation, as demonstrated by MTT assay and caspase-3 activation. TRAIL enhanced mineralized matrix nodule synthesis by C-VICs cultured in osteogenic medium. Conclusions TRAIL is characteristically present within calcific aortic valves, and mediates the calcification of aortic valve interstitial cells in culture through mechanism involving apoptosis.

Liver is a heterogeneous organ central for the control of systemic iron homeostasis. Among the different cell types, only hepatocytes (parenchymal cells) produce the iron regulatory hormone hepcidin. Hepcidin binds to the iron exporter ferroportin and triggers its degradation , thus modulating iron absorption from the diet and iron release from macrophagesdistrib. The iron-mediated hepcidin response is controlled via the SMAD1/5/8 pathway which is activated by bone morphogenetic proteins (BMPs). In contrast to hepcidin, BMPs are mainly produced by liver non-parenchymal cells, questioning the exclusive role of hepatocytes in regulating iron homeostasis. In particular, liver sinusoidal endothelial cells (LSECs) are emerging as cells that sense iron availability and modulate hepcidin levels through the production of BMP2 and BMP6 - the main regulators of hepcidin. However, how LSECs sense iron and which signals control BMP2 and BMP6 production have to be clarified. We have generated a mouse model with heterozygous depletion of the ferroportin allele (Slc40a1wt/trp). These mice show normal haematological parameters, serum iron levels and transferrin saturation. However, the liver is iron deficient and expresses low levels of hepcidin. The phosphorylation of SMAD1/5/8 proteins is lower in Slc40a1wt/trp compared to wild type littermates, explaining the hepcidin phenotype. To further investigate the molecular mechanism underlying hepcidin downregulation mediated by hepatic iron deficiency, we established a protocol to isolate hepatocytes and LSECs from total mouse liver and analyzed expression of iron-related genes. BMP6 is downregulated in LSECs of Slc40a1wt/trp mice, explaining the diminished activity of the SMAD1/5/8 signaling pathway. We next examined intracellular iron levels in hepatocytes and LSECs by measuring ferritin (Ft) and transferrin receptor 1 expression (Tfr1), two genes whose expression is regulated via the IRE/IRP system according to intracellular iron stores. Hepatocytes have an iron-deficient molecular signature, with low levels of Ft and a high expression of Tfr1. Surprisingly in LSECs both Ft and Tfr1 are unchanged, suggesting that this cellular population has unaltered iron levels. Taken together, our results show for the first time that decreased hepatic iron content is self-sufficient to cause a dramatic reduction in hepcidin expression and secretion. The hepcidin downregulation in the Slc40a1wt/trp mouse model is caused by a reduction in BMP6 levels that correlates with hepatocyte iron content. BMP6 modulation cannot be explained either by differences in circulating iron or by altered intracellular iron levels of LSECs, suggesting that BMP6 is not regulated by LSEC-mediated iron sensing. In our mouse model BMP6 seems to be linked with the intracellular iron content of hepatocytes. Therefore, we hypothesize that hepatocytes are the sensor of liver iron content which controls BMP6 expression in LSECs. RNAseq analysis on hepatocytes from Slc40a1wt/trp mice has been performed to identify underlying mechanisms. Disclosures Muckenthaler: Novartis: Research Funding.

Tumor-induced bone disease is a common clinical feature of hematological and metastatic solid cancer. Thus, numerous scientists have gained a better understanding of the mechanisms by which certain tumor types tend to invade specifically the bone. Firstly, Stephen Paget recognized the ‘seed and soil’ hypothesis, stating that cancer cells (the ‘seeds’) can only develop in secondary organs where the microenvironment (the ‘soil’–the bone) is permissive for their growth. Today, this theory has been enlarged to the metastatic process in general, because in order to grow in distant organs, tumor cells need special properties that suit them to those organs. Specifically, in order to metastasize to bone, cancer cells firstly detach from their tissue of origin, subsequently transit through circulation, reside in the bone marrow, and acquire a bone cell-like phenotype responsible for bone establishment and invasion. Each step in the metastatic cascade is rich in biological targets and mechanistic pathways, which are summarized in this review.

Apoptosis can occur throughout the life span of osteoblasts (OBs), beginning from the early stages of differentiation and continuing throughout all stages of their working life. Here, we investigated the effects of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal human OBs showing for the first time that the expression of TRAIL receptors is modulated during OB differentiation. In particular, the TRAIL receptor ratio was in favor of the deaths because of the low expression of DcR2 in undifferentiated OBs, differently it was shifted toward the decoys in differentiated ones. Undifferentiated OBs treated with TRAIL showed reduced cell viability, whereas differentiated OBs displayed TRAIL resistance. The OB sensitiveness to TRAIL was due to the up-regulation of DR5 and the down-regulation of DcR2. The main death receptor involved in TRAIL-reduced OB viability was DR5 as demonstrated by the rescue of cell viability observed in the presence of anti-DR5 neutralizing antibody. Besides the ratio of TRAIL receptors, the sensitivity of undifferentiated OBs to TRAIL-cytotoxic effect was also associated with low mRNA levels of intracellular anti-apoptotic proteins, such as cFLIP, the activation of caspase-8 and -3, as well as the DNA fragmentation. This study suggests that apoptotic effect exerted by TRAIL/TRAIL-receptor system on normal human OB is strictly dependent upon cell differentiation status.

Sclerostin, negative regulator of bone formation, has been originally known as an osteocyte product. Recently, it has been also detected in hypertrophic chondrocytes, distinctive cells of avascular cartilage which is invaded by capillaries and then replaced by vascularized bone. Thus, we hypothesized that sclerostin, in addition to its role already known, may exert an angiogenic activity. We first proved that sclerostin increased the proliferation of human umbilical vein endothelial cells (HUVECs), and next, by using the chicken chorioallantoic membrane (CAM) in vivo assay, we demonstrated that it exerts an angiogenic activity similar to that of vascular endothelial growth factor (VEGF). This last finding was reinforced by several in vitro approaches. Indeed, we showed that sclerostin induced the formation of a network of anastomosing tubules, a significant increase in the percentage of tubule number, total tubule length and number of junctions, as well as the ability of sclerostin-stimulated HUVECs to organize capillary-like structures and closed-meshes similar to VEGF. The angiogenic response elicited by the protein may be due to the binding to its receptor, LRP6, which is highly expressed at mRNA and protein levels by sclerostin treated HUVECs and through the production of two well-known pro-angiogenic cytokines, VEGF and placental growth factor (PlGF). Finally, we demonstrated that sclerostin was also responsible for the recruitment of osteoclasts and their circulating monocyte progenitors. Overall, these findings showed for the first time the new angiogenic in vitro role of sclerostin which could be also considered as a novel molecule in angiogenesis-osteogenesis coupling.

Sclerostin, an osteocyte-expressed negative regulator of bone formation, is one of the inhibitors of Wnt signaling that is a critical pathway in the correct process of osteoblast differentiation. It has been demonstrated that Wnt signaling through the secretion of Wnt inhibitors, such as DKK1, sFRP-2, and sFRP-3, plays a key role in the decreased osteoblast activity associated with multiple myeloma (MM) bone disease. We provide evidence that sclerostin is expressed by myeloma cells that are human myeloma cell lines and plasma cells (CD138(+) cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. Moreover, we show that there are no differences in sclerostin serum levels between MM patients and controls. Thus, our data indicate that MM cells, as a sclerostin source in the BM, could create a microenvironment with high sclerostin concentration that could contribute toward inhibiting osteoblast differentiation.

Dental pulp stem cells (DPSCs) are an adult stem cell population with high proliferative potential and the ability to differentiate in many cell types, and this has led scientists to consider these cells to be an alternative source of postnatal stem cells comparable to mesenchymal stem cells from bone marrow. In this work, we studied the osteoblastic phenotype developed by DPSCs cultured in osteogenic medium. In particular, we analyzed the expression of the typical osteoblast markers such as alkaline phosphatase, collagen type I, osteocalcin, osteopontin, as well as mineralized matrix production. Furthermore, the gene expression during DPSC differentiation into osteoblastic cells was studied by microarray technology. Using microarray and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we found that IGFBP-5, JunB, and NURR1 genes are upregulated during the differentiation of DPSCs. These data indicate that opportunely differentiated DPSCs show a correct osteoblastic phenotype. Therefore, during the osteoblastic differentiation process, IGFBP-5, JunB, and NURR1 gene expression is significantly increased.

Oxytocin (OT) is a primitive neurohypophyseal hormone that plays a primary and indispensible role in mammalian lactation. We have shown recently that OT also regulates bone remodeling, mainly bone formation, with remarkable sensitivity. We now show that OT, apart from its neurohypophyseal origin, is produced in abundance by both human and murine osteoblasts. Production of osteoblast OT is under the control of estrogen, which acts by activating the MAP kinase Erk. This non-genomic mechanism of estrogen action is in stark contrast to its genomic control of OT receptor (OTR) expression. We surmise that there is a local feed-forward loop in bone marrow through which the OT so produced from osteoblasts in response to estrogen acts upon its receptor to exert a potent anabolic action.

There is emerging interest for osteoclasts as key players in the erosive and inflammatory events leading to joint destruction in chronic arthritis. In fact, chronic inflammatory joint diseases such as psoriatic arthritis and rheumatoid arthritis are often characterized by destruction of juxta-articular bone and erosions due to the elevated activity of osteoclasts, which are involved in bone resorption. The main step in inflammatory bone erosion is an imbalance between bone resorption and bone formation: osteoclast formation is enhanced by proinflammatory cytokines such as TNF-α, IL-1β, and IL-17 and is not balanced by increased activity of bone-forming osteoblasts. T-cells, stromal cells, and synoviocytes enhance osteoclast formation via expression of RANKL and, under pathologic conditions, of proinflammatory cytokines. In rheumatoid arthritis, accumulation of osteoclasts in synovial tissues and their activation associated with osteoclastogenic cytokines and chemokines at cartilage erosion sites suggest that they could be usefully selected as therapeutic target. In particular, in consideration of the primary role of RANKL and TNF-α in osteoclastogenesis, the control of the production of RANKL and the inhibition of TNF-α represent important strategies for reducing bone damage in this disease.

Soluble decoy receptor 3 (DcR3), a member of the tumor necrosis factor receptor superfamily, has recently been reported to increase osteoclast (OC) differentiation. Its impact on the skeleton was reinforced by a study on DcR3 transgenic mice showing a decreased bone mass through the elevation of OC number, providing some initial evidence of DcR3 involvement in bone diseases. In this study we show that malignant plasma cells and T lymphocytes from myeloma patients directly produce DcR3, and this molecule supports the elevated formation of OCs in both peripheral blood and bone marrow from the patients. We also show that serum DcR3 levels in myeloma patients are significantly higher compared to controls.

Cells harvested from 12 human giant cell tumors of bone and kept in culture for several passages were characterized for bone-resorbing capability, total and tartrate-resistant acid phosphatase activity, response to the calciotropic hormone calcitonin, cell proliferation, multinucleation after passages, and presence of calcium sensing. Cells obtained from three tumors presented a complete panel of osteoclast characteristics and maintained their multinuclearity after several passages. Cells from four other tumors increased their cAMP levels after treatment with calcitonin, and the other five apparently consisted of cells of stromal origin. These human cell populations with osteoclast characteristics may provide valid in vitro models for the investigation of osteoclastic differentiation and activity.

Multiple myeloma (MM) is a clonal malignancy of terminally differentiated plasma cells. Myeloma patients often have extensive skeletal complications, including bone pain, osteolytic lesions and pathological fractures, which represent the major cause of morbidity and possible mortality. Osteolysis is due to the uncoupling of bone cell activity, caused by osteoclast activation and osteoblast inhibition. Osteoclast biology is dominantly regulated by the RANK/RANKL/OPG axis. A disruption of RANKL/OPG ratio, due to the prevalence of RANKL and/or inactivation of OPG, has been reported in MM bone disease by different mechanisms involving either malignant plasma cells and/or other cells of immune system. Despite the major involvement of RANKL in MM is well documented, a dysregulated production of other cytokines either with pro- or anti-osteoclastogenic activity can also contribute to the development of osteolytic lesions by acting directly on bone cells or altering RANKL/OPG axis. This review focuses on molecules produced by cells of immune system able to induce bone destruction in MM bone disease.

We report that oxytocin (OT), a primitive neurohypophyseal hormone, hitherto thought solely to modulate lactation and social bonding, is a direct regulator of bone mass. Deletion of OT or the OT receptor (Oxtr) in male or female mice causes osteoporosis resulting from reduced bone formation. Consistent with low bone formation, OT stimulates the differentiation of osteoblasts to a mineralizing phenotype by causing the up-regulation of BMP-2, which in turn controls Schnurri-2 and 3 , Osterix , and ATF-4 expression. In contrast, OT has dual effects on the osteoclast. It stimulates osteoclast formation both directly, by activating NF-κB and MAP kinase signaling, and indirectly through the up-regulation of RANK-L. On the other hand, OT inhibits bone resorption by mature osteoclasts by triggering cytosolic Ca 2+ release and NO synthesis. Together, the complementary genetic and pharmacologic approaches reveal OT as a novel anabolic regulator of bone mass, with potential implications for osteoporosis therapy.

Control of osteoclast (OC) apoptosis has been recognized as a critical regulatory factor in bone remodeling. TRAIL, a member of the TNF superfamily, induces apoptosis in neoplastic and normal cells. However, few data are available on the effects of TRAIL on bone cells, thus in the present study we investigated TRAIL role on the apoptosis of human mature OCs. We show that TRAIL treatment causes reduced cell viability, loss of nuclei integrity, and derangement of the actin microfilament in OCs. We also demonstrated that the death receptor DR5, upregulated by TRAIL, could be the mediator of TRAIL-induced OC apoptosis.

It is unclear how physical activity stimulates new bone synthesis. We explored whether irisin, a newly discovered myokine released upon physical activity, displays anabolic actions on the skeleton. Young male mice were injected with vehicle or recombinant irisin (r-irisin) at a low cumulative weekly dose of 100 µg kg(-1). We observed significant increases in cortical bone mass and strength, notably in cortical tissue mineral density, periosteal circumference, polar moment of inertia, and bending strength. This anabolic action was mediated primarily through the stimulation of bone formation, but with parallel notable reductions in osteoclast numbers. The trabecular compartment of the same bones was spared, as were vertebrae from the same mice. Higher irisin doses (3,500 µg kg(-1) per week) cause browning of adipose tissue; this was not seen with low-dose r-irisin. Expectedly, low-dose r-irisin modulated the skeletal genes, Opn and Sost, but not Ucp1 or Pparγ expression in white adipose tissue. In bone marrow stromal cell cultures, r-irisin rapidly phosphorylated Erk, and up-regulated Atf4, Runx2, Osx, Lrp5, β-catenin, Alp, and Col1a1; this is consistent with a direct receptor-mediated action to stimulate osteogenesis. We also noted that, although the irisin precursor Fndc5 was expressed abundantly in skeletal muscle, other sites, such as bone and brain, also expressed Fndc5, albeit at low levels. Furthermore, muscle fibers from r-irisin-injected mice displayed enhanced Fndc5 positivity, and irisin induced Fdnc5 mRNA expression in cultured myoblasts. Our data therefore highlight a previously unknown action of the myokine irisin, which may be the molecular entity responsible for muscle-bone connectivity.

The aim of the present study was to evaluate the effect of bone pate on human osteoblast differentiation by measuring cell viability, alkaline phosphatase activity and expression of the transcription factors and of the major components of the extracellular matrix. Although bone pate has been used in ear surgery for many years and when placed in contact with mastoid and external auditory canal bone become viable, the cellular mechanisms that lead to its osteointegration have never been described. Bone pate taken from four patients subjected to mastoidectomy and affected by middle ear and mastoid cholesteatoma was placed in contact with osteoblast-like cell cultures. Four experimental conditions were obtained: cell cultures treated with bone pate, with bone pate mixed with fibrin glue, with fibrin glue and untreated. After 24 h, the viability of the cells was evaluated; after 1 week, alkaline phosphatase activity and the expression of transcription factors and bone matrix proteins were assessed by quantitative polymerase chain reaction. After 24 h osteoblasts showed increased viability when treated with bone pate (19 % increase) and bone pate mixed with fibrin glue (34 % increase). After 1 week, the number of alkaline phosphatase positive cells increased by 97 and 94 % in cultures treated with bone pate alone and bone pate mixed with fibrin glue. Treatment with bone pate upregulated transcription factors and components of the extracellular matrix. The present data show that bone pate has a high osteoinductive potential on human osteoblasts, enhancing their activity.

Bone has been considered a structure essential for mobility, calcium homeostasis, and hematopoietic function. Recent advances in bone biology have highlighted the importance of skeleton as an endocrine organ which regulates some metabolic pathways, in particular, insulin signaling and glucose tolerance. This review will point out the role of bone as an endocrine “gland” and, specifically, of bone-specific proteins, as the osteocalcin (Ocn), and proteins involved in bone remodeling, as osteoprotegerin, in the regulation of insulin function and glucose metabolism.

CD99 is a transmembrane glycoprotein expressed in physiological conditions by cells of different tissues, including osteoblasts (OBs). High or low CD99 levels have been detected in various pathological conditions, and the supernatant of some carcinoma cell lines can modulate CD99 expression in OB-like cells. In the present work we demonstrate for the first time that two different human myeloma cell lines (H929 and U266) and, in a less degree, their conditioned media significantly downregulate CD99 expression in normal human OBs during the differentiation process. In the same experimental conditions the OBs display a less differentiated phenotype as demonstrated by the decreased expression of RUNX2 and Collagen I. On the contrary, when CD99 was activated by using a specific agonist antibody, the OBs become more active as demonstrated by the upregulation of Alkaline Phosphatase, Collagen I, RUNX2, and JUND expression. Furthermore, we demonstrate that the activation of CD99 is able to induce the phosphorylation of ERK 1/2 and AKT intracellular signal transduction molecules in the OBs.

We report that oxytocin (Oxt) receptors (Oxtrs), on stimulation by the ligand Oxt, translocate into the nucleus of osteoblasts, implicating this process in the action of Oxt on osteoblast maturation. Sequential immunocytochemistry of intact cells or isolated nucleoplasts stripped of the outer nuclear membrane showed progressive nuclear localization of the Oxtr; this nuclear translocation was confirmed by monitoring the movement of Oxtr–EGFP as well as by immunogold labeling. Nuclear Oxtr localization was conclusively shown by Western immunoblotting and MS of nuclear lysate proteins. We found that the passage of Oxtrs into the nucleus was facilitated by successive interactions with β-arrestins (Arrbs), the small GTPase Rab5, importin-β (Kpnb1), and transportin-1 (Tnpo1). siRNA-mediated knockdown of Arrb1, Arrb2, or Tnpo1 abrogated Oxt-induced expression of the osteoblast differentiation genes osterix (Sp7), Atf4, bone sialoprotein (Ibsp), and osteocalcin (Bglap) without affecting Erk phosphorylation. Likewise and again, without affecting pErk, inhibiting Arrb recruitment by mutating Ser rich clusters of the nuclear localization signal to Ala abolished nuclear import and Oxtr-induced gene expression. These studies define a previously unidentified mechanism for Oxtr action on bone and open possibilities for direct transcriptional modulation by nuclear G protein-coupled receptors.

Background: Increased plasma homocysteine (HCY) may be an independent risk factor for osteoporotic fractures and therefore may also adversely affect bone metabolism. We analyzed the effect of HCY on human osteoclast (OC) activity.Methods: We cultured peripheral blood mononuclear cells from 17 healthy male donors [median (SD) age, 30 (5) years] for 20 days with 25 μg/L macrophage-colony-stimulating factor (days 0–11), 20 μg/L receptor-activator of nuclear factor-κB ligand (days 6–20), and 4 different concentrations of HCY (0, 10, 50, and 100 μmol/L; days 0–20). For control purposes, cysteine and glutathione were tested in equimolar concentrations. OCs were identified as large, multinucleated cells with tartrate-resistant acid phosphatase (TRAP) activity and surface vitronectin receptors. We quantified OC activity by measuring TRAP activity. We analyzed cathepsin K (CP-K) activity in 9 donor samples and estimated the dentine-resorbing activity on standard dentine slices in 3 samples.Results: After 20 days of culture, most cells were fully differentiated OCs. TRAP activity increased with increasing HCY concentrations (P Conclusions: Increased HCY concentrations specifically stimulate OC activity in vitro, suggesting a mechanistic role of HCY for bone resorption. Future studies clarifying the mechanistic role of increased HCY concentrations in osteoporosis could have interesting therapeutic implications.

Decreased bone formation contributes to the development of bone lesions in multiple myeloma (MM) patients. In this study, we have investigated the effects of myeloma cells on osteoblast formation and differentiation and the potential role of the critical osteoblast transcription factor RUNX2/CBFA1 (Runt-related transcription factor 2/core-binding factor Runt domain α subunit 1) in the inhibition of osteoblastogenesis in MM. We found that human myeloma cells suppress the formation of human osteoblast progenitors in bone marrow (BM) cultures. Moreover, an inhibitory effect on osteocalcin, alkaline phosphatase, collagen I mRNA, protein expression, and RUNX2/CBFA1 activity by human preosteoblastic cells was observed in cocultures with myeloma cells. The inhibitory effect was more pronounced in the cell-to-cell contact conditions compared with those without the contact and involved the very late antigen 4 (VLA-4) integrin system. Among the soluble osteoblast inhibitors screened, we show the potential contribution of interleukin-7 (IL-7) in the inhibitory effect on osteoblast formation and RUNX2/CBFA1 activity by human myeloma cells in coculture. Finally, our in vitro results were supported in vivo by the finding of a significant reduction in the number of Runx2/Cbfa1-positive cells in the BM biopsies of patients with MM who had osteolytic lesions compared with those who did not have bone lesions, suggesting the critical involvement of RUNX2/CBFA1 in the decreased bone formation in MM. (Blood. 2005;106:2472-2483)

The objective of this study was to evaluate the metabolic in vitro effect of the bisphosphonate neridronate on normal and pathological human osteoblasts. Primary human osteoblast cultures were obtained from cancellous bone of osteoarthritic (OA) and osteoporotic (OP) patients and a corresponding healthy control group. Osteocalcin production was evaluated by cultured cells in neridronate 10(-4) M and 10(-6) M, both under basal conditions and after vitamin D3 stimulation. In the absence of neridronate, vitamin D3 increased osteocalcin production in all cell cultures; under the same conditions, and in the absence of vitamin D3, OA osteoblasts showed a significantly higher osteocalcin production whereas OP osteoblasts showed a significantly lower osteocalcin production compared to the normal osteoblasts, respectively. In all cellular populations neridronate at a higher concentration (10(-4) M) induced a reduction in osteocalcin synthesis, but in normal and osteoarthritic osteoblasts did not reduce the stimulatory effect of vitamin D3, whereas it inhibited the vitamin D3-induced increase of osteocalcin synthesis in the osteoporotic cells. In normal and osteoporotic osteoblasts stimulation with the lower neridronate concentration (10(-6) M) significantly increased osteocalcin production, which was further enhanced by vitamin D3 as an additional effect of the combined treatment. In OA osteoblasts, neridronate 10(-6) M did not induce an increase in osteocalcin synthesis and the additional effect of combined treatment with vitamin D3 was not observed. Neridronate can modify the metabolic activity of human osteoblasts by enhancing or decreasing their biosynthetic activity, both in normal and in pathological conditions, depending on compound concentration and on different cell types. These results confirm the validity of using neridronate at doses usually administered in treating osteoporosis, and they suggest using it to treat other diseases which show an altered osteoblast metabolism, such as osteoarthritis.

The development of multiple myeloma (MM) bone disease is mediated by increased number and activity of osteoclasts (OCs). Using an in vitro osteoclastogenesis model consisting of unstimulated and unfractionated peripheral blood mononuclear cells (PBMCs) from patients with MM, we showed that T cells support the formation of OCs with longer survival. Different from T-cell–depleted MM PBMC cultures, exogenous macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) were necessary for the formation of OCs; however, they did not exhibit longer survival. We found up-regulated production of RANKL, osteoprotegerin (OPG), and TNF-related apoptosis-inducing ligand (TRAIL) by fresh MM T cells. Despite high OPG levels, the persistence of osteoclastogenesis can be related to the formation of the OPG/TRAIL complex demonstrated by immunoprecipitation experiments and the addition of anti-TRAIL antibody which decreases OC formation. OCs overexpressed TRAIL decoy receptor DcR2 in the presence of MM T cells and death receptor DR4 in T-cell–depleted cultures. In addition, increased Bcl-2/Bax (B-cell lymphoma-2/Bcl2-associated protein X) ratio, following Bcl-2 up-regulation, was detected in OCs generated in the presence of T cells. Our results highlight that MM T cells support OC formation and survival, possibly involving OPG/TRAIL interaction and unbalanced OC expression of TRAIL death and decoy receptors.