Adipose tissue is a specialized tissue formed by several depots located below the skin (subcutaneous depots) or in the trunk (visceral depots). It provides the survival of the body by storing/dispensing energy for metabolism and regulating thermogenesis, immune responses, and lactation. Two major types of adipose tissue exist in mammals, the white and brown fats, which are mainly composed of white and brown adipocytes, respectively. White adipocytes primarily act as storage cells for triacylglycerols. They have endocrine functions including the secretion of adipokines such as leptin, resistin, and adiponectin. Brown adipocytes are characterized by a cytoplasm composed of several small lipid droplets and a high amount of mitochondria. They mediate non-shivering thermogenesis, which is the major physiological function of brown adipose tissue. Marrow adipose tissue (MAT) is a third type of adipose tissue, functionally distinct from both white and brown adipose tissues. The development of marrow adipose tissue depends on the stage of life and greatly increases in pathological conditions. Recently, a growing body of evidence has finally confirmed that fully differentiated adipocytes retain the physiological ability to transdifferentiate, undergoing the reprogram of genome and turning into a different cell type with different physiological roles. This peculiar plasticity of adipose tissue ensures that certain physiologic stimuli can induce change of cell phenotype and, consequently, their function. In future, increasingly in-depth analysis of the observed phenomenon of transdifferentiation will contribute to further understanding the plasticity of adipose cells, improving knowledge of their biology and encouraging novel therapeutic strategies.

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.

Scientists have recently focused their attention on adult stem cells as new and more effective treatments for different diseases and disabilities. In fact, it is known that stem cells are capable of renewing themselves and that they can generate multiple cell types. Today, there is new evidence that stem cells are present in far more tissues and organs than once thought and that these cells are capable of developing into more kinds of cells than previously imagined. In this chapter, we focus the attention on teeth as source of stem cells. In particular, we describe the characteristic of the different types of dental stem cells and their use in tissue engineering.

Estrogen uses two mechanisms to exert its effect on the skeleton: it inhibits bone resorption by osteoclasts and, at higher doses, can stimulate bone formation. Although the antiresorptive action of estrogen arises from the inhibition of the MAPK JNK, the mechanism of its effect on the osteoblast remains unclear. Here, we report that the anabolic action of estrogen in mice occurs, at least in part, through oxytocin (OT) produced by osteoblasts in bone marrow. We show that the absence of OT receptors (OTRs) in OTR-/- osteoblasts or attenuation of OTR expression in silenced cells inhibits estrogen-induced osteoblast differentiation, transcription factor up-regulation, and/or OT production in vitro. In vivo, OTR-/- mice, known to have a bone formation defect, fail to display increases in trabecular bone volume, cortical thickness, and bone formation in response to estrogen. Furthermore, osteoblast-specific Col2.3-Cre+/OTRfl/fl mice, but not TRAP-Cre+/OTRfl/fl mice, mimic the OTR-/- phenotype and also fail to respond to estrogen. These data attribute the phenotype of OTR deficiency to an osteoblastic rather than an osteoclastic defect. Physiologically, feed-forward OT release in bone marrow by a rising estrogen concentration may facilitate rapid skeletal recovery during the latter phases of lactation.

Differentiation therapy is an attractive treatment for osteosarcoma (OS). CD99 is a cell surface molecule expressed in mesenchymal stem cells and osteoblasts that is maintained during osteoblast differentiation while lost in OS. Herein, we show that whenever OS cells regain CD99, they become prone to reactivate the terminal differentiation program. In differentiating conditions, CD99-transfected OS cells express osteocyte markers, halt proliferation, and largely die by apoptosis, resembling the fate of mature osteoblasts. CD99 induces ERK activation, increasing its membrane-bound/cytoplasmic form rather than affecting its nuclear localization. Through cytoplasmic ERK, CD99 promotes activity of the main osteogenic transcriptional factors AP1 and RUNX2, which in turn enhance osteocalcin and p21WAF1/CIP1, leading to G 0/G1 arrest. These data underscore the alternative positions of active ERK into distinct subcellular compartments as key events for determining OS fate.

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 differentiatedDPSCs show a correct osteoblastic phenotype. Therefore, during the osteoblastic differentiation process, IGFBP-5, JunB, andNURR1gene expression is significantly increased

Abstract There are conflicting data regarding the potential impact of chronic glucocorticoid (GC) therapy on the bone mineral density of patients with congenital adrenal hyperplasia (CAH). Previous studies performed by dual-energy X-ray absorptiometry reported conflicting results. The purpose of this study was to assess the impact of chronic GC replacement treatment in children with classical and non classical CAH due to 21-hydroxylase deficiency (21-OHD) by quantitative ultrasonometry (QUS), an easy, cheap, and radiation-free technique. The study population consisted of nineteen 21-OHD patients (nine males) on lifelong GC treatment. Anthropometric, hormonal, and treatment data were recorded for each patient, and bone quality was assessed by QUS measurements. QUS findings (amplitude-dependent speed of sound and bone transmission time) were normal in 21-OHD patients and did not correlate with duration of treatment, daily, total, and yearly hydrocortisone dose. Furthermore, no significant correlation was found between QUS findings and 17α-hydroxy progesterone, Δ4-androstenedione, and testosterone levels. In conclusion, our results provide reassurance that currently used replacement doses of GC do not have a major impact on bone in patients with CAH. QUS seems to be a reliable tool for screening of bone health in children with 21-OHD

T cells may affect the activity of osteoclasts, the bone resorbing cells. In particular, recently authors focalize their attention on CD8+ T-cells, demonstrating that murine pre-osteoclasts can recruit naïve CD8+ T cells and induce them to differentiate in regulatory cytotoxic T cells (TcREG), which in turn may suppress osteoclast formation and activity. Thus, here we describe the methodological approach needed to obtain TcREG and to evaluate TcREG effects on murine osteoclast activity and formation in an in vitro experimental model.

Prior studies show that oxytocin (Oxt) and vasopressin (Avp) have opposing actions on the skeleton exerted through high-affinity G protein-coupled receptors. We explored whether Avp and Oxtr can share their receptors in the regulation of bone formation by osteoblasts. We show that the Avp receptor 1α (Avpr1α) and the Oxt receptor (Oxtr) have opposing effects on bone mass: Oxtr(-/-) mice have osteopenia, and Avpr1α(-/-) mice display a high bone mass phenotype. More notably, this high bone mass phenotype is reversed by the deletion of Oxtr in Oxtr(-/-):Avpr1α(-/-) double-mutant mice. However, although Oxtr is not indispensable for Avp action in inhibiting osteoblastogenesis and gene expression, Avp-stimulated gene expression is inhibited when the Oxtr is deleted in Avpr1α(-/-) cells. In contrast, Oxt does not interact with Avprs in vivo in a model of lactation-induced bone loss in which Oxt levels are high. Immunofluorescence microscopy of isolated nucleoplasts and Western blotting and MALDI-TOF of nuclear extracts show that Avp triggers Avpr1α localization to the nucleus. Finally, a specific Avpr2 inhibitor, tolvaptan, does not affect bone formation or bone mass, suggesting that Avpr2, which primarily functions in the kidney, does not have a significant role in bone remodeling.

21-Hydroxylase deficiency (21-OHD) is the most common cause of congenital adrenal hyperplasia (CAH), resulting from deletions or mutations of the P450 21-hydroxylase gene (CYP21A2). Children with 21-OHD need chronic glucocorticoid (cGC) therapy, both to replace congenital deficit in cortisol synthesis and to reduce androgen secretion by adrenal cortex. GC-induced osteoporosis (GIO) is the most common form of secondary osteoporosis that results in an early, transient increase in bone resorption accompanied by a decrease in bone formation, maintained for the duration of GC therapy. Despite the conflicting results in the literature about the bone status on GC-treated patients with 21-OHD, many reports consider these subjects to be at risk for osteoporosis and fractures. In bone cells, at the molecular level, GCs regulate various functions including osteoblastogenesis, osteoclastogenesis, and the apoptosis of osteoblasts and osteocytes. In this paper, we focus on the physiology and biosynthesis of endogenous steroid hormones as well as on the effects of GCs on bone cells, highlighting the pathogenetic mechanism of GIO in children with 21-OHD.

Children with 21-hydroxylase deficiency (21-OHD) need chronic glucocorticoid (cGC) therapy to replace congenital deficit of cortisol synthesis and this therapy is the most frequent and severe form of drug-induced-osteoporosis. We found in 21-OHD patients high serum and leukocyte levels of dickkopf-1 (DKK1), a secreted antagonist of the Wnt/β-catenin signaling pathway, known to be a key regulator of bone mass. In particular, we demonstrated by flow cytometry, confocal microscopy and real time PCR that monocytes, T lymphocytes and neutrophils from patients expressed high levels of DKK1, which may be related to the cGC therapy. In fact, we showed that dexamethasone treatment markedly induced the expression of DKK1 in a dose- and time-dependent manner in leukocytes. The serum from patients containing elevated levels of DKK1 can directly inhibit in vitro osteoblast differentiation and RANKL expression. We also found a correlation between both DKK1 and Receptor Activator of NF-kappaB Ligand (RANKL) or C-terminal telopeptides of Type I collagen (CTX) serum levels in 21-OHD patients on cGC treatment. Our data indicated that DKK1, produced by leukocytes, may contribute to the alteration of bone remodeling in 21-OHD patients on cGC treatment.

Studies have given new insights into the pathogenesis of myeloma bone disease and demonstrated a strong interdependence between the immune and skeletal system as well as tumor growth and osteoclast activity. Discovery of the receptor activator of the nuclear factor kB (RANK)/RANK ligand (RANKL)system brought a rapid progress in the understanding of the regulatory mechanism of osteoclast differentiation in physiological and pathological conditions. However, numerous connections have been discovered in osteoimmunology beyond merely the actions of RANKL. These include both cells and cytokines participating in osteoclast development and bone destruction. The resulting bone destruction releases several cytokines which in turn promote myeloma cell growth, thus maintaining a vicious circle between the bone destructive process and tumor progression. Therefore, the inhibition of bone resorption could not only decrease myeloma bone disease, but also the tumor progression. Furthermore, research into osteoimmunology promises the discovery of new strategies and the development of innovative therapeutics to cure or alleviate bone loss in multiple myeloma bone disease

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 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 to 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-/- . 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. This article is protected by copyright. All rights reserved.

Irisin, a novel myokine produced in response to physical exercise by skeletal muscle, displays anabolic effect on bone and can improve the bone-loss-induced osteoporosis in hind limb suspended mice. It is well known that muscles positively impact the skeleton and in different sports, including soccer, total body bone mineral density (TB-BMD) is elevated. Therefore, we have investigated the correlation between irisin serum levels and total and bone sub-regional BMD in soccer players never studied before. In this study, Caucasian football players of Bari team have been enrolled. Their sera were collected to measure by ELISA kit irisin levels and by dual-energy X-ray absorptiometry (DEXA) analysis measurements of BMD (g • cm−2) in the whole body and different bone sub-regions (head, arms, legs, ribs, dorsal vertebrae, lumbar vertebrae, pelvis) were performed. The BMC (g) was measured in the whole body. By means of Pearson’s (R) and Cohen’s (d) coefficient we investigated the linear association between the irisin serum levels and BMD. In soccer players, we have found a positive correlation between irisin and TB-BMD as demonstrated by the values of Pearson and Cohen’s (d) coefficient. Furthermore, linear association was detected between irisin and BMD of different bone-site such as right arm, lumbar vertebrae and head. A positive trend was also observed analyzing circulating levels of irisin and bone mineral content as well as total Z-score. In conclusion, we have demonstrated the correlation between irisin and total or bone sub-regional BMD in soccer players for the first time, an additional systemic effect of the “sport-hormone” defined myokine.

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 differentiation in vitro to 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.

Irisin, a hormone-like myokine, regulates energy homeostasis and mediates the benefits of physical activity on health.

It has been recently reported that, after physical exercise activity, the skeletal muscle releases Irisin, the newly identified myokine able of driving transition of white adipocytes into brown, following a phenomenon known as the browning response. This result suggested that skeletal muscle is crucial in the regulation of energy homeostasis, supporting its role as endocrine organ that targets adipose tissue by promoting energy expenditure. In accordance with this new finding, we demonstrated that conditioned media (CM) collected from primary myoblasts of exercised mice were able to induce osteoblast differentiation in a greater extent than those of mice housed in resting conditions and this effect is Irisin-mediated. In view of further proving the involvement of Irisin in bone metabolism, we validate its direct effect on osteoblasts by using r-Irisin. Here we show that phosphorylation of MAP kinase ERK and expression of Atf 4 (p<0,001), the key trascription factor of osteoblast differentiation, were significantly increased after Irisin treatment. Furthermore, ALP and pro-Collagen I mRNA resulted up regulated (p<0,001), as we already demonstrated by treating osteoblasts with conditioned medium from primary myoblasts of exercised mice. To recapitulate in vivo the effect of physical exercise, we injected mice with r-Irisin. Our results show that BV/TV of Irisin-treated mice was higher than vehicle-injected mice. In elderly, the severe decline of skeletal muscle function, known as Sarcopenia, is associated with impaired function of bone (Osteopenia) and these two simultaneous losses of function lead to increased risk of bone fractures. In order to reveal new strategies for treatment of sarcopenia and osteopenia, we also analyzed the effect of physical activity in old mice. Our findings demonstrate that mRNA levels of the most relevant bone proteins resulted up regulated in ex-vivo osteoblast obtained from exercised old mice compared with mice kept in resting conditions. Our data highlight a novel link in muscle-fat-bone axis demonstrating that Irisin targets bone tissue directly. Future perspectives, based on these studies, could satisfy the ongoing research of exercise-mimetic therapies with anabolic action on the skeleton

Background: Multiple myeloma (MM)-osteolytic bone disease occurs in about 70% of newly diagnosed patients, and up to 90% at relapse. It is characterized by an unbalanced bone remodelling, due to increased osteoclast (OC) activity and impaired osteoblast repair. Osteoclastogenesis is regulated by members of TNF superfamily. Among these, there is LIGHT/TNFSF14 recently implicated in the pathogenesis of joint diseases with increased bone resorption, as the rheumatoid arthritis. LIGHT is expressed by activated T-cells, monocytes, granulocytes, spleen cells, and immature dendritic cells, and it is described as a potent T-cell co-stimulatory molecule. Aims: Here, we purposed to investigate a possible role of LIGHT in the mechanisms of the enhanced osteoclastogenesis occurring in MM-osteolytic bone disease. Methods: Peripheral blood (PB) and bone marrow (BM) aspirates were obtained from 40 patients (23M/17F, median age: 64 years), newly diagnosed as having symptomatic MM with or without osteolytic bone disease, smoldering MM (sMM) or Monoclonal Gammopathy of Undetermined Significance (M.G.U.S.). Osteolytic lesions were detected by skeleton standard radiography, and in some cases also by spine and pelvis nuclear magnetic resonance. The control group included PB and BM aspirates from 15 patients with nonneoplastic disease without any skeletal involvement as well as PB from 25 healthy donors matching for age and sex with the patients’ group. Patients and controls gave their written informed consent to the study, approved by the local Ethical Committee and performed according to the Declaration of Helsinki. By means of flow cytometry, Western Blotting and real-time PCR, LIGHT expression was evaluated in freshly purified CD14+ monocytes, CD2+ T-cells and neutrophils from PB and BM aspirates of patients and controls. OCs were obtained from unfractionated PB mononuclear cells (PBMCs) cultured in the presence or in the absence of an anti-LIGHT neutralizing monoclonal antibody (mAb). Mature OCs were identified as multinucleated tartrate-resistant acid phosphatase (TRAP) positive cells. Results: In the CD14+ monocytes, CD2+ T-cells and neutrophils isolated from PB and BM of patients with MM-osteolytic bone disease, at both protein and mRNA levels LIGHT was found more expressed than in the cells isolated from the patients with symptomatic MM without bone disease, sMM, M.G.U.S, nonneoplastic disease without any skeletal involvement, and healthy donors. The in vitro effect of the anti-LIGHT mAb on osteoclastogenesis resulted in a significant reduction of the OC formation (p<0.001). Summary and Conclusions: Our findings support a possible involvement of LIGHT in the mechanisms of the osteoclastogenesis occurring in MM-osteolytic bone disease.

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 RANKL, osteoprotegerin, sclerostin, DKK1, 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 were inside the normal range, but femoral-BMD T-score reached osteopenic levels. Consistently, in AKU adults higher RANKL and CTX and lower osteoprotegerin levels were observed than 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 TNFα, 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 which appears in adult AKU.

A significant number of long-term complications have been described in childhood leukemia survivors. In particular, these patients may present features of metabolic syndrome (MetS), and therefore increased risk for cardiovascular diseases. The aim of this meta-analysis is to evaluate the prevalence and the risk of MetS in survivors of childhood leukemia. Two authors independently performed a systematic literature search in PubMed and EMBASE to March 2014, reviewed and selected articles, based on pre-determined selection criteria. Twelve articles, comprising 2,337 participants (1,462 cases and 875 controls), were included in the meta-analysis. Only three of them were case–control studies eligible for the meta-analysis. The childhood leukemia survivors showed an increased risk of MetS as compared to healthy controls (OR = 4.36; 95 % CI 1.19–16.22). The risk was significantly increased only in patients treated with chemotherapy and radiotherapy (OR = 7.79; 95 % CI 1.27–47.77), and not in patients treated with only chemotherapy (OR = 2.35; 95 % CI 0.40–13.78). Childhood leukemia survivors, in particular if treated also with radiotherapy, are prone to develop MetS more than healthy controls. Monitoring of MetS components in these patients is necessary to avoid cardiovascular consequences later in life.

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. Areas covered: 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. Expert opinion: 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.

Wingless-type (Wnt) signaling through the secretion of Wnt inhibitors Dickkopf1, soluble frizzled-related protein-2 and -3 has a key role in the decreased osteoblast (OB) activity associated with multiple myeloma (MM) bone disease. We provide evidence that another Wnt antagonist, sclerostin, an osteocyte-expressed negative regulator of bone formation, is expressed by myeloma cells, that is, human myeloma cell lines (HMCLs) and plasma cells (CD138+ cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. We demonstrated that BM stromal cells (BMSCs), differentiated into OBs and co-cultured with HMCLs showed, compared with BMSCs alone, reduced expression of major osteoblastic-specific proteins, decreased mineralized nodule formation and attenuated the expression of members of the activator protein 1 transcription factor family (Fra-1, Fra-2 and Jun-D). Moreover, in the same co-culture system, the addition of neutralizing anti-sclerostin antibodies restored OB functions by inducing nuclear accumulation of β-catenin. We further demonstrated that the upregulation of receptor activator of nuclear factor κ-B ligand and the downregulation of osteoprotegerin in OBs were also sclerostin mediated. Our data indicated that sclerostin secretion by myeloma cells contribute to the suppression of bone formation in the osteolytic bone disease associated to MM.

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.

Apoptosis can occur throughout the entire 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 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 favour 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.

Background: Stem cells are defined as clonogenic cells capable of self-renewal and multi-lineage differentiation. A population of these cells has been identified in human Dental Follicle (DF). Dental Follicle Stem Cells (DFSCs) were found in pediatric unerupted wisdom teeth and have been shown to differentiate, under particular conditions, into various cell types of the mesenchymal tissues. Aim: The aim of this study was to investigate if cells isolated from DF show stem features, differentiate toward osteoblastic phenotype and express osteoblastic markers. Methods: We studied the immunophenotype of DFSCs by flow cytometric analysis, the osteoblastic markers of differentiated DFSCs were assayed by histochemical methods and real-time PCR. Results: We demonstrated that DFSCs expressed a heterogeneous assortment of makers associated with stemness. Moreover DFSCs differentiated into osteoblast-like cells, producing mineralized matrix nodules and expressed the typical osteoblastic markers, Alkaline Phosphatase (ALP) and Collagen I (Coll I). Conclusion: This study suggests that DFSCs may provide a cell source for tissue engineering of bone.

Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal dysplasia characterized by hypoplastic or aplastic clavicles, dental abnormalities, and delayed closure of the cranial sutures. In addition, mid-face hypoplasia, short stature, skeletal anomalies and osteoporosis are common. We aimed to evaluate osteoclastogenesis in a child (4 years old), who presented with clinical signs of CCD and who have been diagnosed as affected by deletion of RUNX2, master gene in osteoblast differentiation, but also affecting T cell development and indirectly osteoclastogenesis. The results of this study may help to understand whether in this disease is present an alteration in the bone-resorptive cells, the osteoclasts (OCs). Unfractionated and T cell-depleted Peripheral Blood Mononuclear Cells (PBMCs) from patient were cultured in presence/absence of recombinant human M-CSF and RANKL. At the end of the culture period, OCs only developed following the addition of M-CSF and RANKL. Moreover, real-time PCR experiment showed that freshly isolated T cells expressed the osteoclastogenic cytokines (RANKL and TNFα) at very low level, as in controls. This is in accordance with results arising from flow cytometry experiments demonstrating an high percentage of circulating CD4(+)CD28(+) and CD4(+)CD27(+) T cells, not able to produce osteoclastogenic cytokines. Also RANKL, OPG and CTX serum levels in CCD patient are similar to controls, whereas QUS measurements showed an osteoporotic status (BTT-Z score -3.09) in the patient. In conclusions, our findings suggest that the heterozygous deletion of RUNX2 in this CCD patient did not alter the osteoclastogenic potential of PBMCs in vitro.

Stem cells are a promising tool for bone tissue regeneration. Dental pulp stem cells (DPSCs) can be easily obtained even in human young adults. In this study we investigated the capability of DPSCs, to express the osteoblastic phenotype when cultured with osteogenic medium. DPSCs isolated from the dental pulp of impacted third molar teeth were cultured with appropriate medium to induce osteoblast differentiation. Using Western-Blot, RT-PCR and microarray analysis, we studied the expression of osteoblastic parameter, and by Von Kossa staining we evaluated the production of mineralized matrix nodules. The results were compared with controls represented by undifferentiated DPSCs. DPSCs, differentiated into osteoblast-like cells, express large amount of alkaline phosphatase (ALP), collagen I (Coll I), osteopontin (OPN) and osteocalcin (OCN), all these parameters characterizing the osteoblastic phenotype. Differentiated DPSCs express Runx2 and JunB, a member of the AP-1 complex; both the transcription factors are associated with osteoblast differentiation and skeletal morphogenesis. Moreover, DPSCs express insulin growth factor-binding protein 5 (IGFBP-5), one of the regulating proteins of IGFs function. Finally, DPSCs can form mineralized matrix nodules that are a feature exclusive to osteoblasts. DPSCs could represent a potential source of osteoblasts to be used for bone regeneration.

Studies concerning the pathophysiological connection between obesity and osteoporosis are currently an intriguing area of research. Although the onset of these two diseases can occur in a different way, recent studies have shown that obesity and osteoporosis share common genetic and environmental factors. Despite being a risk factor for health, obesity has traditionally been considered positive to bone because of beneficial effect of mechanical loading, exerted by high body mass, on bone formation. However, contrasting studies have not achieved a clear consensus, suggesting instead that excessive fat mass derived from obesity condition may not protect against osteoporosis or, even worse, could be rather detrimental to bone. On the other hand, it is hitherto better established that, since adipocytes and osteoblasts are derived from a common mesenchymal stem cell precursor, molecules that lead to osteoblastogenesis inhibit adipogenesis and vice versa. Here we will discuss the role of the key molecules regulating adipocytes and osteoblasts differentiation, which are peroxisome proliferators activated receptor-γ and Wnts, respectively. In particular, we will focus on the role of both canonical and non-canonical Wnt signalling, involved in mesenchymal cell fate regulation. Moreover, at present there are no experimental data that relate any influence of the Wnt inhibitor Sclerostin to adipogenesis, although it is well known its role on bone metabolism. In addition, the most common pathological condition in which there is a simultaneous increase of adiposity and decrease of bone mass is menopause. Given that postmenopausal women have high Sclerostin level inversely associated with circulating estradiol level and since the sex hormone replacement therapy has proved to be effective in attenuating bone loss and reversing menopause-related obesity, we hypothesize that Sclerostin contribution in adipogenesis could be an active focus of research in the coming years.

Bone loss and fractures are consequences of aging, diseases or traumas. Furthermore the increased number of aged people, due to the rise of life expectancy, needs more strategies to limit the bone loss and regenerate the lost tissue, ameliorating the life quality of patients. A great interest for non-pharmacological therapies based on natural compounds is emerging and focusing on the oligostilbene Polydatin, present in many kinds of fruits and vegetables, when resveratrol particularly in red wines. These molecules have been extensively studied due to their antioxidant and anti-inflammatory effects, showing more recently Resveratrol the ability to enhance osteogenic differentiation and bone formation. However, the clinical applications of Resveratrol are limited due to its low bioavailability and rapid metabolism, while its natural glycosilated precursor Polydatin shows better metabolic stability and major abundance in fresh fruits and vegetables. Nevertheless the role of Polydatin on osteogenic differentiation is still unexplored. Mesenchymal stem cells (MSCs) from dental tissues, such as dental bud stem cells (DBSCs), are able to differentiate toward osteogenic lineage: thus we investigated how Resveratrol and Polydatin influence the differentiation of DBSCs, eventually affecting bone formation. Our results showed that Polydatin increases MSCs osteogenic differentiation sharing similar properties with Resveratrol. These results encourage to deepen the effects of this molecule on bone health and its associated mechanisms of action, wishing for the future a successful use in bone loss prevention and therapy.

Although hyponatremia is known to be associated with osteoporosis and a high fracture risk, the mechanism through which bone loss ensues has remained unclear. As hyponatremic patients have elevated circulating arginine-vasopressin (AVP) levels, we examined whether AVP can affect the skeleton directly as yet another component of the pituitary-bone axis. Here, we report that the two Avp receptors, Avpr1α and Avpr2, coupled to Erk activation, are expressed in osteoblasts and osteoclasts. AVP injected into wild-type mice enhanced and reduced, respectively, the formation of bone-resorbing osteoclasts and bone-forming osteoblasts. Conversely, the exposure of osteoblast precursors to Avpr1α or Avpr2 antagonists, namely SR49059 or ADAM, increased osteoblastogenesis, as did the genetic deletion of Avpr1α. In contrast, osteoclast formation and bone resorption were both reduced in Avpr1α-/- cultures. This process increased bone formation and reduced resorption resulted in a profound enhancement of bone mass in Avpr1α-/- mice and in wild-type mice injected with SR49059. Collectively, the data not only establish a primary role for Avp signaling in bone mass regulation, but also call for further studies on the skeletal actions of Avpr inhibitors used commonly in hyponatremic patients.

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.

Bone has been considered as 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.

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.

LIGHT (TNFSF14), expressed by different cells of the immune system, binds two trans-membrane receptors: HVEM and LTβR. It is over-expressed in erosive rheumatoid arthritis and lytic myeloma-bone disease and controversial data have been published on its role osteoclast (OC) formation in vitro. Here, we investigated the role of LIGHT on in vitro murine osteoclastogenesis model and bone phenotype in LIGHT−/− mice. Firstly, we showed that murine macrophages stimulated with LIGHT alone did not differentiate into OCs. Consistently, the addition of agonist anti-HVEM and anti-LTβR antibodies did not affect osteoclastogenesis in the same cultures. Interestingly, the presence of LIGHT and sub-optimal RANKL concentration displayed synergic effects on OC formation through the early and sustained activation of Akt, NFκB and JNK pathways. Secondly, by microCT we found that the femurs of LIGHT−/− mice exhibited a 30% (P<0.01) decrease in trabecular BV/TV due to a significant reduction in trabecular thickness and number as well as the increase in trabecular spaces respect to WT mice. Furthermore, a fivefold increase of OC number/bone surface was found in femora from KO mice compared to WT (P<0.004). To investigate the possible molecular mechanism/s responsible for this bone phenotype in LIGHT−/− mice we studied OPG levels in whole bone marrow (BM) extracts from the femurs of these mice and demonstrated a significant reduction in OPG mRNA transcript respect to WT (fourfold, P<0.001). Further investigations showed that BM CD8+ T cells and B cell subpopulations from KO mice expressed lower levels of OPG compared to those from WT mice. Consistently, LIGHT treatment in a dose dependent manner increase OPG expression in BM CD8+ T cells and B-cells. In conclusion, our results identified LIGHT as a new important modulator of bone remodeling and highlighted a new modulator of OPG expression.

LIGHT, expressed by different cells of the immune system, binds two trans-membrane receptors: HVEM and LT?R. It is over-expressed in erosive rheumatoid arthritis and lytic myeloma-bone disease and controversial data have been published on its role in vitro osteoclast (OC) formation. Here, we investigated the role of LIGHT on in vitro murine osteoclastogenesis model and bone phenotype in LIGHT-/- mice. Firstly, we showed that murine macrophages stimulated with LIGHT alone did not differentiate into OCs. Consistently, the addition of agonist anti-HVEM and anti-LT?R antibodies did not affect osteoclastogenesis in the same cultures. Interestingly, the presence of LIGHT and sub-optimal RANKL concentration displayed synergic effects on OC formation through the early and sustained activation of Akt, NF?B and JNK pathways. Secondly, by microCT we found that the femurs of LIGHT-/- mice exhibited a decrease in trabecular BV/TV due to a significant reduction in trabecular thickness and number as well as the increase in trabecular spaces respect to wild-type (WT) mice. Further, a deformity of the vertebral column due to the collapse of vertebral bodies was radiographically observed in about 40% of LIGHT-/- mice. To investigate the possible molecular mechanism/s responsible for this bone phenotype in LIGHT-/- mice we studied OPG levels in whole bone marrow extracts from the femurs of these mice and demonstrated a significant reduction in OPG mRNA transcript respect to WT. Based on the knowledge that mature B cells are the major source of OPG and that they represent about 50% of the spleen cells, we treated with LIGHT total splenocytes demonstrating a three fold increase of OPG mRNA levels respect to the untreated cells. In conclusion, our results suggested that the net effect of LIGHT on bone remodeling probably occurs through the modulation of OPG expression. However, further experiments are needed to better clarify this issue

Calcific aortic valve disease (CAVD) represents a slowly progressive pathologic process associated with major morbidity and mortality. The process is characterized by multiple steps: inflammation, fibrosis, and calcification. Numerous studies focalised on its physiopathology highlighting different “actors” for the multiple “acts”. This review focuses on the role of the tumor necrosis factor superfamily (TNFSF) members in the pathogenesis of CAVD. In particular we discuss on clinical and experimental studies providing evidence of the involvement of tumor necrosis factor alpha (TNF-α), receptor activator of nuclear factor kappa-B (NF-kB) ligand (RANKL), its membrane receptor RANK and its decoy receptor osteoprotegerin (OPG), and TNF-related apoptosis-inducing ligand (TRAIL) in valvular calcification.

Glucocorticoid induced osteoporosis (GIO) is the most frequent form of drug induced osteoporosis. Glucocorticoids (GCs) affect osteoblastogenesis, osteoclastogenesis, and promote the apoptosis of osteoblasts and osteocytes. A decrease of bone mineral density (BMD) has been described in several pediatric diseases that require GCs, both as long term replacement therapy, such as Congenital Adrenal Hyperplasia, and as treatment of acute phase or relapses, such as asthma, juvenile rheumatoid arthritis, inflammatory bowel diseases, systemic lupus erythematosus, organ transplantation, and Steroid Sensitive Nephrotic Syndrome. The increasing number of children with GIO and at risk of fractures reflects the complex nature of this condition, and the need of development of anti-osteoporotic drugs. In this review, we focus on the mechanisms of GIO in some pediatric diseases and on treatment of osteoporosis. We also report data on new signaling pathways as potential targets for future anti-osteoporotic drugs.

Il nostro brevetto verte sul possibile impiego di Irisina, molecola fisiologica prodotta dal muscolo durante l'esercizio, come farmaco in grado di prevenire e curare l'osteoporosi. Tale patologia è caratterizzata da una riduzione della densità minerale ossea (BMD) e da un concomitante deterioramento della massa muscolare scheletrica (Sarcopenia) che porta ad un aumento del rischio di frattura. Dai nostri risultati è emerso che la somministrazione di Irisina, in modelli sperimentali murini osteoporotici, è in grado sia di prevenire che curare osteoporosi e sarcopenia. L'Irisina agisce su tutte le popolazioni di cellule presenti nell'osso ma risulta particolarmente attiva sugli osteoblasti, cellule deputate alla formazione di nuovo osso, rendendo tali cellule più numerose e più attive nella produzione di nuova matrice ossea. Questo si traduce in aumento della densità minerale ossea (BMD) e rende l'osso più resistente alle fratture. Tale effetto è ulteriormente potenziato dall'azione che irisina esercita sul muscolo scheletrico, il cui deterioramento è sempre associato allo sviluppo di osteoporosi. La somministrazione di irisina, infatti, nei nostri modelli sperimentali, che sviluppavano osteoporosi e sarcopenia, si è dimostrata capace di prevenire entrambe le patologie. Pertanto, visti gli effetti che irisina è in grado di esercitare contemporaneamente sullo scheletro e sul muscolo, ne consegue che lo sviluppo di un farmaco a base di irisina potrebbe essere un efficace supporto non solo per la cura ma anche per la prevenzione dell'osteoporosi e della sarcopenia. Questo porterebbe ad una riduzione del rischio di frattura con conseguente riduzione della spesa del Sistema Sanitario Nazionale. Inoltre, l'impiego di irisina potrebbe essere fortemente competitivo rispetto ai farmaci attualmente impiegati per la cura dell’osteoporosi, che seppure di comprovata efficacia presentano una serie di limitazioni ed effetti collaterali. L'impiego di irisina potrebbe: 1) non presentare gli effetti collaterali tipici dei farmaci al momento disponibili in quanto molecola fisiologica; 2) essere utilizzata anche in fase preventiva quando vi sono fattori di rischio a sviluppare osteoporosi; 3) essere utilizzata per prevenire e curare al contempo osteoporosi e sarcopenia in un colpo solo.

Object of the present invention is the use of Irisin for the treatment and/or prevention of osteoporosis. In particular, the present invention refers to the use of recombinant irisin for the treatment and/or prevention of osteoporosis.