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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.

Occupancy of the chicken osteoclast alpha v beta 3 integrin stimulates immediate cell signals. Peptides from osteopontin containing Arg-Gly-Asp and peptides from the osteopontin and bone sialoprotein sequences containing Arg-Gly-Asp stimulated immediate reductions in osteoclast cytosolic Ca2+. The changes in cytosolic Ca2+ required the Arg-Gly-Asp sequence, and were blocked by LM609, a monoclonal antibody to the alpha v beta 3 integrin. Osteoclast stimulation by the proteins through the integrin did not require immobilization since soluble peptides produced changes in cytosolic Ca2+ and inhibited osteoclast binding to bone particles and bone resorption. The decrease in cytosolic Ca2+ stimulated by osteopontin and related peptides was due to activation of a plasma membrane Ca(2+)-ATPase. Thus, the data suggest that ligand binding to the osteoclast alpha v beta 3 integrin results in a reduction in cytosolic Ca2+ which participates in regulation of osteoclast function.

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.

Publisher Summary 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.

Decoy receptor 3 (DcR3), a member of the tumor necrosis factor (TNF) receptor superfamily, is known to be involved in cell survival and osteoclast (OC) formation. In this study, we show that malignant plasma cells and T lymphocytes from multiple myeloma (MM) bone disease patients, as well as Karpas 909, a human myeloma cell line, directly produce DcR3. By interacting with FasL, this molecule could inhibit OC apoptosis. In fact, the use of a neutralizing anti-DcR3 antibody induces a reduction of cell viability with a consequent increase of apoptotic cell number, the activation of caspase-8 and -3, and DNA fragmentation. Furthermore, we show that DcR3 supports OC formation in samples from MM patients through the upregulation of RANKL and TNFalpha by T lymphocytes and only TNFalpha by CD14+ cells. In conclusion, our data provide the first evidence of the expression of DcR3 in MM, and the involvement of this molecule in supporting the survival and formation of OCs from MM bone disease patients. The production of DcR3 by T lymphocytes confers these cells a role in the pathogenesis of bone disease associated with MM.

During space flight, severe losses of bone mass are observed. Both bone formation and resorption are probably involved, but their relative importance remains unclear. The purpose of this research is to understand the role of osteoclasts and their precursors in microgravity-induced bone loss. Three experiments on isolated osteoclasts (OCs) and on their precursors, OSTEO, OCLAST, and PITS, were launched in the FOTON-M3 mission. The OSTEO experiment was conducted for 10 d in microgravity within bioreactors with a perfusion system, where the differentiation of precursors, cultured on a synthetic 3-dimensional bonelike biomaterial, skelite, toward mature OCs was assessed. In OCLAST and in PITS experiments, differentiated OCs were cultured on devitalized bovine bone slices for 4 d in microgravity. All of the experiments were replicated on ground in the same bioreactors, and OCLAST also had an inflight centrifuge as a control. Gene expression in microgravity, compared with ground controls, demonstrated a severalfold increase in genes involved in osteoclast maturation and activity. Increased bone resorption, proved by an increased amount of collagen telopeptides released VS ground and centrifuge control, was also found. These results indicate for the first time osteoclasts and their precursors as direct targets for microgravity and mechanical forces.

In order to examine the effects of vitamin D on osteoblast function and to evaluate if osteoporotic and normal osteoblasts show a different behaviour in response to vitamin D, this report investigates the changes in osteocalcin production, after 1,25-dihydroxy-vitamin D(3) stimulation of cultured osteoblasts derived from osteoporotic patients. Our results indicate an inadequate osteoblastic function in osteoporosis and demostrate that 1,25-dihydroxy-vitamin D(3) can stimulate the metabolic activity of human osteoblasts in vitro. Considering that osteoporotic bone samples were representative of senile osteoporosis, our results may indicate a different metabolic phenotype in osteoporotic osteoblasts compared with normal osteoblasts. The increased osteocalcin production after 1,25-dihydroxy-vitamin D(3) stimulation of osteoporotic osteoblasts suggests a reduced, but not absent, anabolic function in senile osteoporotic osteoblasts. The results of this study confirm the validity of vitamin D(3) to treat senile osteoporosis and suggest the need of higher vitamin D(3) intake in senile osteoporotic patients than in younger subjects.

Aggressive bone resorption is well recognized in psoriatic arthritis (PsA). By using in vitro osteoclastogenesis models, consisting of unstimulated mononuclear cells from peripheral blood (PBMCs) and synovial fluid (SFMCs) of PsA patients, we demonstrated the spontaneous OC formation in both culture systems. In PBMCs the osteoclastogenesis was T cell-dependent while it was T cell- and fibroblast-dependent in SFMCs. T cells isolated from PBMCs and SFMCs of PsA patients overexpressed RANKL and TNF-alpha, and fibroblasts from SFMCs overexpressed RANKL. Thus, T cells and fibroblasts from synovial fluid through the production of osteoclastogenic cytokines could support the OC formation in PsA patients.

Anabolic skeletal agents have recently broadened the therapeutic options for osteoporosis by directly stimulating bone formation and improving bone turnover, bone density, bone size, and bone microarchitecture. We recently demonstrated that two new L: -carnitine derivatives, L: -carnitine fumarate (LC) and isovaleryl-L: -carnitine fumarate (Iso-V-LC), stimulated osteoblast proliferation and differentiation. We here investigated, by histomorphometry in a mouse model of osteoporosis, the impact of these compounds on the repair of trabecular bone and the osteoblast involvement in this process. Fifty-nine inbred adult female CD1 mice in pregnancy were assigned to four treatment groups: (1) controls, mice fed a standard normocalcemic pre- and postpartal diet; (2) Hypo, mice fed a low-calcium isocaloric prepartal diet and a standard postpartal diet; (3) LC, mice fed a group 2-type diet supplemented post-partum with LC; (4) Iso-V-LC, mice fed a group 2-type diet supplemented post-partum with Iso-V-LC. Bone volume/total volume ratio (BV/TV), bone perimeter, osteoblast surface/bone surface, and osteoblast number/bone surface were measured from sections of L3 and L4 vertebral bodies obtained from animals killed on the day of delivery (controls and Hypo) and on days 7, 14, and 21 after delivery (all groups). BV/TV and all osteoblast-based indexes were significantly higher in LC and Iso-V-LC than in Hypo mice at each time point, and Iso-V-LC at the end of the treatment attained levels observed in controls. In conclusion, Iso-V-LC and, to a lesser extent, LC accelerated the recovery of normal BV/TV level after a hypocalcemic diet.

The number and activity of osteoclasts (OCs) are critical for maintaining normal bone turnover. The number is determined by the rates of cell differentiation and death. TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces apoptosis by interacting with its death receptors, (DR4, DR5). However, its activity can be modulated by two decoy receptors, (DcR1 and DcR2). In this paper we show that TRAIL treatment causes reduced OC viability as well as an increased apoptotic OC number. Loss of nuclei integrity and derangement of the actin microfilament were also induced by TRAIL in OCs. Moreover, we demonstrated the expression of all TRAIL receptors in both precursors and differentiated OCs, and the upregulation of DR5 during OC differentiation. Interestingly, DcR2 was upregulated in the early stage of osteoclastogenesis and downregulated at the end of the differentiation process. We showed that DR5, upregulated by TRAIL, could be the mediator of TRAIL-induced OC apoptosis, since the addition of anti-DR5 neutralizing antibodies restores the OC viability previously reduced by TRAIL. Furthermore, the intracellular pathway induced by TRAIL in OCs involves caspase-8 and Bid activation. In conclusion, our data highlight an important role for the TRAIL/TRAIL receptor system in the regulation of OC apoptosis.

Psoriatic arthritis (PsA) is an inflammatory joint disease, characterized by extensive bone resorption, whose mechanisms have not been fully elucidated. Thus, in the present study we investigated the involvement of RANKL, TNFalpha, and IL-7 in the osteoclastogenesis of PsA patients. In vitro osteoclastogenesis models, consisting of unfractionated and T-cell-depleted mononuclear cells from peripheral blood (PBMCs) and synovial fluid (SFMCs) of 20 PsA patients as well as from healthy donors were studied. Freshly isolated T and B cells from PBMCs and T cells and fibroblasts from SFMCs of PsA patients were subjected to RT-PCR to detect the levels of RANKL, TNFalpha, and IL-7. Osteoclastogenesis was studied in the presence of RANK-Fc, anti-TNFalpha, and anti IL-7 functional antibodies. We demonstrate that lymphocytes and fibroblasts support osteoclast (OC) formation in PsA patients through the production of osteoclastogenic cytokines. In particular, OC formation was completely abolished in unstimulated T cell-depleted PBMC cultures, and reduced by approximately 70% in unstimulated T cell-depleted SFMC cultures. Freshly isolated T cells from PBMCs and SFMCs of PsA patients overexpressed RANKL and TNFalpha, while fibroblasts from synovial fluid produced only RANKL. We show that the presence of RANK-Fc and/or anti-TNFalpha functional antibodies reduced OC formation. Moreover, T and B cells from PBMCs as well as T cells and fibroblasts from SFMCs expressed IL-7 mRNA. Finally, the anti-IL-7 functional antibody significantly reduced osteoclastogenesis. Our results suggest that fibroblasts, B and T lymphocytes support OC formation by producing RANKL, TNFalpha, and IL-7, contributing to the aggressive bone resorption in PsA patients.

Background Interleukin-7 (IL-7) is a potent regulator of lymphocyte development, which has also significant effects on bone; in fact it is a potent osteoclastogenic factor. Some human solid tumors produce high IL-7 levels, suggesting a potential IL-7 role on tumor development and progression. Methodology We studied 50 male patients affected by solid tumors, and their blood samples were collected at tumor diagnosis. PBMCs were isolated and cultured with/without IL-7 to study its influence on osteoclastogenesis. Serum and cell culture supernatant IL-7 levels were measured by ELISA. The quantitative analysis of IL-7 expression on T and B cells was performed by Real-Time PCR. Principal findings Serum IL-7 levels were highest in osteolytic cancer patients, followed by cancer patients without bone lesions, and then healthy controls. We showed the IL-7 production in PBMC cultures and particularly in monocyte and B cell co-cultures. A quantitative analysis of IL-7 expression in T and B cells confirmed that B cells had a high IL-7 expression. In all cell culture conditions, IL-7 significantly increased osteoclastogenesis and an anti-IL-7 antibody inhibited it. We demonstrated that IL-7 supports OC formation by inducing the TNF-alpha production and low RANKL levels, which synergize in promoting osteoclastogenesis. Conclusions We demonstrated the presence of high serum IL-7 levels in patients with bone metastasis, suggesting the use of serum IL-7 level as a clinical marker of disease progression and of bone involvement. Moreover, we showed the capability of IL-7 to stimulate spontaneous osteoclastogenesis of bone metastatic patients and to induce osteoclastogenesis in cancer patients without bone involvement. These findings add further details to the disclosure of the mechanisms controlling bone metastasis in solid tumors.

Multiple myeloma (MM) is often associated with an increased osteoclast (OC) activity. Using an in vitro osteoclastogenesis model consisting of MM unstimulated and unfractionated peripheral blood mononuclear cells (PBMCs), we showed that T cells support OC formation and survival. Differently, in T cell-depleted MM PBMC cultures, exogenous macrophage-colony stimulating factor (M-CSF) and receptor activated of nuclear factor-kappaB ligand (RANKL) were necessary for osteoclastogenesis. We found RANKL, OPG, and TRAIL overexpression by fresh MM T cells. Despite high osteoprotegerin (OPG) levels, the persistence of osteoclastogenesis can be related to the formation of the OPG/TRAIL complex. Our results highlight that MM T cells support OC formation and survival, possibly involving OPG/TRAIL interaction.

Background. The TNF superfamily member LIGHT, known to play a major role in T-cell homeostasis, has been reported in erosive bone disease associated with rheumatoid arthritis. Herein, we investigated whether LIGHT is implicated in the mechanisms leading to Multiple Myeloma (MM)-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 bone disease, smoldering MM (sMM) or M.G.U.S. Bone disease assessment was performed by skeleton x-Ray, and spine and pelvis NMR or CT. The control group included PB and BM aspirates from 15 patients with non-neoplastic disease without any skeletal involvement, and PB from 25 healthy-donors matching for age and sex with the MM group. Patients and controls gave their written informed consent to the study, approved by Ethical Committee of University Hospital of Bari, and performed according to Declaration of Helsinki. By means of flow cytometry, western blotting, and real-time PCR, LIGHT expression was assessed in freshly purified CD14+ monocytes, CD2+ T-cells and neutrophils from PB and BM aspirates of patients and controls. Osteoclasts (OCs) were obtained from unfractionated PBMC cultures treated or not with an anti-LIGHT neutralizing monoclonal antibody (mAb). Mature OCs were identified as multinucleated tartrate-resistant acid phosphatase (TRAP) positive cells. In cultures from BM mononuclear cells (BMNCs), the formation of CFU-F and CFU-OB was evaluated in the presence or absence of anti-LIGHT neutralizing mAb. CFU-F and CFU-OB were identified with alkaline phosphatase (ALP) or Von Kossa staining, respectively. Further, in CFU-F and CFU-OB cultures, the expression of OB differentiation markers was analyzed by real-time PCR. Results. We found overexpression of LIGHT on CD14+ monocytes, CD8+ T-cells and neutrophils of PB and BM from MM-bone disease patients, in whom LIGHT induced osteoclastogenesis and inhibited osteoblastogenesis, as we demonstrated by culture treatment with an anti-LIGHT antibody. Moreover, in cultures from healthy-donors, we found that LIGHT induced osteoclastogenesis in RANKL-dependent and –independent manners. In particular, in the presence of a sub-optimal RANKL concentration, LIGHT and RANKL showed synergic effects on osteoclast formation, associated to early and sustained activation of Akt, NFκB and JNK pathways. Otherwise in cultures of BM samples from patients without bone disease, LIGHT treatment inhibited the formation of CFU-F and CFU-OB, and the expression of osteoblastic markers such as collagen-I, osteocalcin and bone sialoprotein-II, supporting a LIGHT indirect inhibition of osteoblastogenesis, possibly and to some extent, through sclerostin expression by monocytes. Conclusions. Our findings, for the first time, provide evidence that LIGHT plays a role in MM-bone disease development by increasing osteoclastogenesis and decreasing osteoblastogenesis. Disclosures No relevant conflicts of interest to declare.

In recent years, many procedures based on surface modification have been suggested to improve the biocompatibility and biofunctionality of orthopedic titanium-based implants. In this contest, the development of a new titanium-based biomaterial that could be covalently modified with biologically active molecules (i.e., RGD-peptides, growth factors, etc.) able to improve osteoblasts response was investigated. The strategy followed was based on a preliminary coating of the implant material by an adherent thin polymer film to which bioactive molecules could be grafted exploiting the polymer surface chemical reactivity. In this work, we focused our attention on pyrrole-3-acetic acid (Py-3-acetic), a pyrrole with carboxylic acid substituent, whose electrosynthesis and characterization on titanium substrates were already accomplished and whose potentialities in the design of new biocompatible surfaces are well evident. As first step, the biocompatibility of the electrochemically grown PPy-3-acetic films was investigated performing in vitro tests (adhesion and proliferation) with mouse bone marrow cells. Successively, the availability and reactivity of surface carboxylic groups were tested through the grafting of an aminoacidic residue to PPy-3-acetic films.

Periodontitis is characterized by alveolar bone destruction; however, the mechanisms responsible for bone damage are poorly understood. It has been reported that T cells are implicated in the pathogenesis of periodontitis. It has been also demonstrated that activated T lymphocytes secrete receptor activator of nuclear factor-kappa B ligand (RANKL) and can support the differentiation of monocytes into resorbing osteoclasts (OCs). Therefore, the purpose of this study was to examine the OC formation in periodontitis patients (PP) and the role of T cells in osteoclastogenesis.To study OC formation, we used an in vitro model consisting of unstimulated and unfractionated peripheral blood mononuclear cells (PBMCs) from PP and controls. In parallel, T-cell-depleted PBMCs from the same patients were also established. The expression of RANKL and tumor necrosis factor-alpha (TNF-alpha) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot in fresh T cells isolated from PP and controls. Functional antibodies, anti-RANKL and anti-TNF-alpha, were utilized to study osteoclastogenesis in PBMC cultures from PP.We showed that, in unfractionated PBMCs from PP, the OCs spontaneously developed in a T-cell-dependent way. The addition of macrophage colony stimulating factor (MCSF) and RANKL was necessary to promote the osteoclastogenesis in T-cell-depleted PBMC cultures from PP and in unfractionated PBMCs from periodontally healthy controls. Moreover, freshly isolated T cells from PBMCs of PP overexpressed RANKL and TNF-alpha. Finally, functional anti-RANKL and anti-TNF-alpha antibodies significantly inhibited osteoclastogenesis.Our data suggest that T cells support spontaneous osteoclastogenesis in PP via RANKL and TNF-alpha overexpression.

We studied the biological features and the immunophenotype of a cell culture established from the lesion of soft tissues of a woman affected by Gorham-Stout syndrome. We found that these cells belonged to a monocytic lineage with some characteristics of immature osteoclasts and were able to release large amounts of osteoclastogenic and angiogenic molecules that may contribute to disease progression. Introduction: Gorham-Stout syndrome is a rare disease characterized by osteolysis and proliferation of vascular or lymphatic vessels, with a severe outcome. Its etiology and the identification of the cell types involved are completely unknown. Materials and Methods: A cell culture from a lesion of soft tissues was established, and its behavior in vitro and in immunodeficient mice was studied. We analyzed (1) the cell phenotype by flow cytometry; (2) the adhesive and migratory properties on different substrates; (3) the ability to differentiate into mature osteoclasts; (4) the production of osteclastogenic and angiogenic molecules; (5) the in vivo angiogenic activity of the cells subcutaneously implanted in mouse in a Matrigel plug; and (6) the ability to recapitulate the disease when transplanted in nude mice. Results and Conclusions: The established culture consisted of a morphologically homogeneous cell population belonging to a monocytic lineage having some features of an osteoclast-like cell type. Cells had an invasive phenotype, were angiogenic, and produced osteoclastogenic (IL-6, TGF-β1, IL-1β) and angiogenic (vascular endothelial growth factor-A {VEGF-A}, CXCL-8) molecules when challenged with inflammatory cytokines. Immunodeficient mice injected with these cells did not show any bone lesions or vascular alteration, but had high amounts of circulating human IL-6 and VEGF-A. Cells isolated from a cutaneous lymphangiomatosis did not show any of these findings. These data suggest that cells of monocyte-macrophage lineage play an essential role in the pathogenesis of Gorham-Stout disease, whose progression is propelled by cytokine circuits that accelerate angiogenesis and osteoclastogenesis.

Age-related bone loss is characterized by decreased osteoblast activity, possibly related to the reduction of energy production. Carnitine promotes energy availability and its concentration declines with age; Therefore, two Carnitine derivatives, L-carnitine fumarate (LC) and isovaleryl L-carnitine fumarate (Iso-V-LC), have been tested on several parameters of human osteoblasts in vitro. Both compounds significantly increased osteoblast activity, but the new compound Iso-V-LC was more efficient than LC at lower concentrations. They both significantly enhanced cell proliferation, [3H]-proline incorporation and the expression of collagen type I (COLLI), and the bone sialoproteins (BSPs) and osteopontin (OPN). The percentage of alkaline phosphatase (ALP)-positive cells and the secretion of osteocalcin were not modified by LC and Iso-V-LC. Both molecules increased the formation of mineralized nodules, but Iso-V-LC reached the maximum effect at a concentration 10-fold lower than that of LC. Furthermore, we showed that insulin-like growth factor (IGF)-I and IGF-II mRNA levels were not modified by the treatment. However, the two compounds induced an increase of insulin-like growth factor binding protein (IGFBP)-3 and a decrease of IGFBP-5 in both osteoblast lysates and the extracellular matrix (ECM). In conclusion these data suggest that carnitine and, in particular, its new derivative, Iso-V-LC supplementation in the elderly may stimulate osteoblast activity and decrease age-related bone loss.

Increasing evidences demonstrated many new targets for the hypothalamic hormone oxytocin, as the regulation of food balance and in some cases of leptin secretion. Considering that leptin is a potent inhibitor of bone formation and that oxytocin receptors (OTR) were detected in normal human osteoblasts, we investigated if OTR was expressed by human osteoclasts (hOCs) and the effect of the hormone on these cells. Here, we demonstrate by immunofluorescence and by Western blot analysis the expression of OTR by fully differentiated hOCs and by their precursors (pOCs). We also show that the receptor is functional, as OT treatment induces an increase of [Ca2+]i, and that the hormone may affect osteoclastogenesis, since it increases the number of pre-osteoclasts.

Meningoencephalic herniations into the middle ear and mastoid are rare and histologic information has been supplied only in few reports. Sections from two idiopathic encephaloceles were compared with those from encephaloceles associated with previous trauma, infection or surgery described in the literature. In the idiopathic encephaloceles, the herniated tissue consisted of cortical neural tissue with degenerative changes and gliosis like that described in cases with a history of chronic infection, previous surgery or trauma, but no inflammatory cells were present.

Breast cancer is a common disease in females but very rare in males, in whom it shows a more metastatic behavior, and a worse prognosis. Matrix metalloprotease-2 (MMP-2) and MMP-9 are proteolytic enzymes balanced by tissue inhibitor of MMP-2 (TIMP-2), commonly involved in cancer metastasis. This is the first study on gelatinolytic activity in male breast cancer patients, compared to that in female patients. In cancer tissues, both gelatinases were more expressed than in normal samples, being and more concentrated in male than in female patients. TIMP-2 levels were slightly increased in normal compared to those in cancer tissues and more concentrated in males than in females. Immunostaining showed that in male cancer tissues MMP-2 and MMP-9 staining was more intense and diffuse than in female cancer tissues, while no differences were observed regarding TIMP-2. In conclusion, the increased expression of gelatinases in male breast cancer patients together with anatomical features might explain the high tendency toward metastasis and the worse prognosis.

Integrins are membrane receptors that mediate interactions between cells and the extracellular matrix. We recently showed that the osteoclast integrin alpha(v)beta(3) exists in two different conformations, so-called 'basal' and 'activated', with each exhibiting a distinct function. In this study we demonstrate that, in non-resorbing osteoclasts, the 'activated' form of alpha(v)beta(3) accumulates in the motile areas of the plasma membrane. During bone resorption this conformation is prevalent in the ruffled membrane, whereas the 'basal' form of alpha(v)beta(3) is also present in the sealing zone. Moreover, hepatocyte growth factor (HGF) and macrophage colony stimulating factor (M-CSF), two molecules involved in osteoclastogenesis and osteoclast survival, modulate alpha(v)beta(3) conformation in vitro. Preincubation with HGF or M-CSF induces a shift of conformation of alpha(v)beta(3) in primary human osteoclasts (OCs) and in the osteoclast-like cell line (GCT 23). Activated integrin promotes osteoclast migration to the alpha(v)beta(3) ligand osteopontin and enhances bone resorption. Thus, HGF and M-CSF modulate the alpha(v)beta(3) conformational states required for osteoclast polarization and resorption. The capacity of growth factors to alter the affinity of alpha(v)beta(3) toward its ligands offers a potential explanation for the diverse responses of osteoclasts to the same ligand.

Chronic kidney disease (CKD) is a progressive loss in renal function, which severity is classified in five stages, with stage 1 being the mildest and stage 5 being a severe illness evolving to hemodialysis (HD). Chronic inflammatory state in HD and CKD patients (pts) may contribute to the development of bone disease through the increase of osteoclasts (OCs), the bone resorbing cells. In pathological condition, immune cells and inflammatory cytokines belonging to tumor necrosis factor superfamily, such as LIGHT and RANKL, play a key role in osteoclastogenesis. The aim of our study was to evaluate the osteoclastogenic potential of unfractionated and T cell-depleted PBMCs from CKD and HD pts. We also evaluated the presence of circulating osteoclast precursors (CD14+/CD16+) and characterized the immune cell subsets for LIGHT and RANKL expression through flow cytometry and RT-PCR. PBMCs from HD and CKD pts (stage IV-V) showed a spontaneous osteoclastogenesis in vitro (80±7). Conversely, exogenous cytokines were essential to trigger and sustain osteoclastogenesis in CKD pts (stage I-II) and controls (10±4). It correlated with a significant increase of both LIGHT and RANKL expression on CD4+ T cells as well as CD14+CD16+ monocytes in HD and CKD pts (stage IV-V) compared to controls (p

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.

In this research we utilized tail-suspended rats as an in vivo model for bone loss studies in order to investigate the effects of the tail suspension on the structure of the suspended bones and in ex vivo cultures the activities of trabecular osteoblasts, marrow-derived osteogenic cells, and osteoclasts obtained from treated animals, compared with untreated controls. After a 5-day hind limb unloading, trabecular thinning was already evidenced in the tibial primary spongiosa. In the secondary spongiosa, the bone formation activity was reduced whereas osteoclastic parameters were not yet altered. Bone marrow-derived osteogenic cells and differentiated osteoblasts from enzymatic digestion of posterior limb trabecular bone were prepared from 5 day tail-suspended rats and from normally loaded rats as controls. Cell morphology, alkaline phosphatase (ALPH) activity, production of mineral matrix, osteocalcin, and IL-6 secretion were evaluated in both cell populations. Tail suspension reduced the osteogenic potential of stromal marrow cells and of already differentiated osteoblasts. In fact, ALP positive colonies were significantly reduced in number and were smaller in size compared with controls and bone nodules formed in permissive conditions were also significantly fewer and smaller, whereas in cultures of cells from control conditions, large mineralizing nodules were formed. Osteocalcin secretion was not affected by unloading. Finally, IL-6 concentration was increased in marrow-derived cells from treated rats compared with controls. Primary cultures of osteoclasts were obtained from the nonadherent fraction of the bone marrow of the same animals. The number of TRAP positive cells in culture from tail-suspended rats was significantly increased, as well as bone resorption activity, measured as resorbed surfaces of a suitable synthetic hydroxyapatite, compared with controls. These data clearly suggest that skeletal unloading not only reduces the osteogenic potential of osteoblastic cells but induces an increased osteoclastogenesis and osteoclast activity in ex vivo cultures. They also indicate for the first time that a possible mediator responsible for the increased osteoclastogenesis could be represented by the IL-6 whose secretion by bone marrow cells was significantly enhanced by unloading.

The covalent attachment of an Arg-Gly-Asp (RGD) containing peptide to polypyrrole(PPy)-coated titanium substrates has been investigated in order to develop a bioactive material of potential use in orthopedic fields. Polypyrrole has been employed as the coating polymer because of its suitability to be electrochemically grown directly onto metallic substrates of different shapes, leading to remarkably adherent films. The synthetic peptide Cys-Gly-(Arg-Gly-Asp)-Ser-Pro-Lys, containing the cell-adhesive region of fibronectin (RGD), has been grafted to the polymer substrate via the cysteine residue using a procedure recently developed in the authors laboratory. The effectiveness of grafting was monitored by X-ray photoelectron spectroscopy (XPS), which assessed the presence of the peptide grafted onto the polymer surface exploiting the cysteine sulfur as target element. Neonatal rat calvarial osteoblasts were attached to RGD-modified PPy-coated Ti substrates at levels significantly greater than on unmodified PPy-coated Ti and glass coverslip substrates.

Introduction Multiple myeloma (MM)-bone disease occurs in 70% to 80% of patients at MM diagnosis, and up to 90% at relapse; skeletal related events cause high morbidity and mortality. MM-bone disease is mostly consisting of lytic lesions arising as a consequence of an unbalanced bone remodelling due to activation of osteoclasts (OCs), and inactivation of osteoblasts. Osteoclastogenesis may be under immunoregulation by T-cells through the production of receptor activator of NF-kB ligand (RANKL), that is the OC differentiating molecule. Aim By means of an in vitro osteoclastogenesis model derived from peripheral blood mononuclear cells (PBMCs) of patients with MM-lytic bone disease, we purposed to evaluate OC formation and T-cell RANKL expression at diagnosis, and in the setting of frontline therapy. Methods PB was taken by venipuncture from 18 patients (7 men and 11 women), aged from 63 to 84, diagnosed as having symptomatic MM by International Myeloma Working Group (IMWG) criteria. In all of them, lytic lesions were demonstrated by skeleton standard radiography, and in some cases by nuclear magnetic resonance (NMR) too. On the basis of patients’ age and fitness, initial standard therapy consisting of proteasome inhibitor (Bortezomib) associated with Melphalan and Prednisone (VMP) was administered for nine cycles. The therapy response was assessed according to IMWG criteria. The controls included PB collected from 20 healthy donors, matched for age and sex with the patients. Controls and patients gave their written informed consent to the study, that was approved by the local Ethical Committee and performed according to the Declaration of Helsinki. PB was taken from the patients at diagnosis, and following the fourth and the ninth VMP cycles, respectively. OCs were obtained from unfractionated PBMC cultures either stimulated or not with the exogenous pro-osteoclastogenic cytokines [macrophage-colony stimulating factor (M-CSF) and RANKL]. Mature OCs were identified as multinucleated tartrate-resistant acid phosphatase (TRAP) positive cells. Freshly isolated T-cells from patients’ and controls’ PBMCs were lysed and analyzed by Western Blotting to evaluate RANKL expression. Results Spontaneous osteoclastogenesis was demonstrated in the cultures derived from PBMCs isolated at diagnosis. With particular regard to the patients achieving a deep treatment response, at the end of the culture period we found that PBMCs formed few small-sized OCs, when cultured in the absence of M-CSF and RANKL. Moreover, fresh T-cells purified after four and nine VMP cycles expressed progressively lower RANKL levels than fresh T-cells purified at diagnosis. Otherwise the cultures derived from non-responsive patients’ PBMCs, collected during and after treatment respectively, showed in vitro spontaneous formation of numerous and large OCs, overlapping the results at diagnosis. Fresh T-cells purified during or after treatment expressed comparable or even higher RANKL levels respect to diagnosis. Conclusions These findings show that in vitro spontaneous osteoclastogenesis and T-cell RANKL expression may be correlated with the response to treatment in patients with MM-lytic bone disease. However, further studies will be necessary to confirm these data, and to better translate them from a biologic to a clinical point of view. Disclosures: No relevant conflicts of interest to declare.

Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption, but the mechanism responsible for tumor-mediated osteoclast activation has not yet been clarified. In the present study we utilized a well-known human breast cancer cell line (MDA-231) in order to assess its capability to influence osteoclastogenesis in human bone marrow cultures and bone resorption in fully differentiated osteoclasts. We demonstrated that conditioned medium (CM) harvested from MDA-231 increased the formation of multinucleated TRAP-positive cells in bone marrow cultures. Bone resorption activity of fully differentiated human osteoclasts and of osteoclast-like cell lines, from giant cell tumors of bone (GCT), was highly increased by the presence of MDA-231 CM. Moreover, while MDA-231 by themselves did not produce IL-6 tumor cell, CM increased the secretion of IL-6 by primary human osteoclasts and GCT cell lines compared to untreated controls. These data suggest that MDA-231 produce osteoclastic activating factor(s) that increase both osteoclast formation in bone marrow culture and bone resorption activity by mature cells. Moreover, breast cancer cells stimulate IL-6 secretion by osteoclasts that is one of the factors known to supports osteoclastogenesis.

Publisher Summary This chapter discusses the osteoclast cytoskeleton. Eukaryotic cells contain three major classes of cytoskeletal fibers: 7 nm diameter actin microfilaments, 24 nm diamenter microtubules (MTs), and 10 nm diameter intermediate filaments. All of these fibers are formed by finely regulated polymerization of protein subunits. These cytoskeletal elements play a fundamental role not only in determining the steady-state organization of the cytoplasm, but also in facilitating selective delivery between spatially segregated organelles. The actin and MT cytoskeletal systems are closely interrelated both structurally and functionally in cells. Both filament systems commonly coexist within domains of the cytoplasm, and factors that cause redistribution of one often lead to a change in the distribution of the other. Cell motility is a cytoskeleton-dependent function. At the leading edge of migrating mouse fibroblasts, a biased assembly of actin is involved in the formation and stabilization of protrusions, while the perinuclear tail region exhibits a net disassembly of actin fibers. The osteoclast can be in a secretory or in a migratory state depending on the phase of its activity. The more recent findings on cytoskeleton involvement in these processes can very possibly apply to this very peculiar cell. Podosomes are highly dynamic and can assemble and disassemble in minutes. They are also linked to integrin receptors, endowed with the capability of linking several proteins of the extracellular matrix. The number and distribution of podosomes is related to osteoclastic resorptive activity. During bone resorption, they are thickly packed in a ring-like structure that surrounds the resorption lacuna, so closely assembled to resemble, at low magnification, a continuous actin ring. In nonresorbing osteoclasts, they can be observed in small groups scattered on the ventral membrane, or in ring-like fashion, but singularly identifiable.

Hepatocyte growth factor (HGF), also known as scatter factor, is a powerful motogen, mitogen, and morphogen produced by cells of mesodermal origin, acting on epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c-MET protooncogene. We show that the HGF receptor is expressed by human primary osteoclasts, by osteoclast-like cell lines, and by osteoblasts. In both cell lineages, HGF stimulation triggers the receptor kinase activity and autophosphorylation. In osteoclasts, HGF receptor activation is followed by increase in intracellular Ca2+ concentration and by activation of the pp60c-Src kinase. HGF induces changes in osteoclast shape and stimulates chemotactic migration and DNA replication. Osteoblasts respond to HGF by entering the cell cycle, as indicated by stimulation of DNA synthesis. Interestingly, osteoclasts were found to synthesize and secrete biologically active HGF. These data strongly suggest the possibility of an autocrine regulation of the osteoclast by HGF and a paracrine regulation of the osteoblast by the HGF produced by the osteoclast.

e13525 Background: Notch signalling is deregulated in a number of cancers and is possibly involved in cancer stem cell development. It is postulated that cancers such as breast can become addicted to Notch signalling presenting opportunities for tumour-specific intervention. Due to the presence of at least 4 cell surface receptors that mediate Notch signalling, conventional targeting agents such as monoclonal antibodies cannot inhibit them all. As all the signalling cascades converge on the Notch transcriptional complex (NTC) in the nucleus, inhibition of gene expression by disrupting this machinery is an attractive strategy to overcome this redundancy. Methods: Towards this, we have developed a fusion protein based on the potent cell penetrating protein (CPP) antennapedia (Antp) recombinantly fused to a dominant-negative truncated mastermind-like protein (DN-MAML) called TR4. We have also developed a method of incorporating CPP to deliver intracellular antibody fragments against nuclear targets such as Notch and mastermind. Results: The Antp-DN-MAML protein (TR4) has similar potency to many of the gamma-secretase inhibitors being developed against this pathway but more specific for the NTC.TR4 can inhibit tumour cells in vitro and in vivo and provide some insight into the mechanism of action. Also such intracellular targets are accessible to antibodies, provided they are delivered using CPP such as antennapedia. Conclusions: .As a model system, scFvs have been raised to key epitopes in the NTC which can lead to complex inhibition and the same downstream effects as TR4.In addition, we further demonstrate that there is a more general approach to trascription factor inhibition using single-chain Fvs fused to Antp

It has been widely demonstrated that the Notch receptor signaling pathway is required for regulation and differentiation of stem cells in many tissues cell types. It is also well established that aberrant Notch signaling can lead to several human forms of cancer, including leukaemia, breast, colon, skin, cervical, and lung cancer, making Notch an attractive target for new cancer therapies. It has become clear that one of the major therapeutic targets in the Notch pathway is the cleavage of four Notch receptors, in which gamma-secretase inhibitors play a role. Unfortunately inhibition of γ-secretase complex is accompanied by many serious side effects. In order to avoid the side effects associated with γ-secretase inhibitors and achieve an improved therapeutic effect, a more selective Notch inhibition is desirable. We have genetically engineered a fusion protein, consisting of the Drosophila transcription factor Antennapedia (ANTP) and the truncated version of Mastermind-like (MAML) that behaves in a dominant negative (DN) fashion and inhibits the Notch transactivation complex. This fusion protein, (ANTP/DN-MAML) or TR4, has been tested for its ability to target tumor cells in vitro and in vivo. Our results indicate that targeting Notch signaling with the ANTP/DN-MAML (TR4) suppresses Notch activation, translocates into nucleus and inhibits tumor growth in mice, without significant organ or systemic toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4364. doi:10.1158/1538-7445.AM2011-4364

We tested whether recognition of bone-related peptides regulates intracellular Ca2+ concentration ([Ca2+]i) of giant cell tumor of bone (GCT). [Ca2+]i was measured in single cells by fura 2 fluorometry. GCT cells were sensitive to bone sialoprotein-II (BSP-II), osteopontin (OPN), and related fragments. Responses consisted of a prompt increase of [Ca2+]i, mostly transient, with a peak followed by a rapid return toward baseline. Responses were not mimicked by bovine plasma fibronectin. Sensitivity of GCT cells to bone peptides was specific, since BALB/3T3 fibroblasts and U-937 histiocytic lymphoma cells with monocytic phenotype failed to respond to BSP-II and OPN fragments. GRGDSP synthetic esapeptide, carrying the Arg-Gly-Asp adhesive motif, and GRGESP (Asp replaced by Glu), but not the GRADSP (Gly replaced by Ala), were active in inducing [Ca2+]i transients as well. Responses were observed also in cells treated with the BSP-II 1C fragment, lacking any known adhesive sequence, indicating that the active peptides inducing [Ca2+]i increments may be multiple. Sensitivity to extracellular matrix peptides was present in a variable fraction of the cells and was downregulated on long-term culture. The mechanism inducing [Ca2+]i elevations was mostly related to Ca2+ release from thapsigargin-sensitive intracellular pools.

Abstract 2961 Reduced osteoblast activity contributes to the development of multiple myeloma-bone disease. Wingless-type (Wnt) signalling pathway is critical in osteoblastogenesis, and it is negatively regulated by molecules such as frizzled-related proteins (sFRPs), Dickkopf proteins (DKKs) and sclerostin. Myeloma cells are known to induce inhibition of osteoblastogenesis through Wnt antagonists such as DKK-1 and sFRP-2 and -3 whereas the role of sclerostin, an osteocyte-expressed negative regulator of bone formation, has not been yet investigated. We provide novel evidence showing sclerostin expression by myeloma cells from patients with multiple myeloma-bone disease and human myeloma cell lines (HMCLs). By means of a co-culture system of bone marrow stromal cells (BMSCs) and HMCLs, we demonstrated that sclerostin expression by myeloma cells and HMCLs is responsible for reduced expression of major osteoblastic specific proteins namely bone-specific alkaline phosphatase, collagen-type I, bone sialoprotein II and osteocalcin as well as decreased mineralized nodule formation and attenuated expression of member of the AP-1 transcription factor family (i.e. Fra-1, Fra-2 and Jun-D). The addition of a neutralizing anti-sclerostin antibody to our co-culture system can restore the above parameters, through the intranuclear accumulation of β-catenin in BMSCs. On the other hand, we demonstrated that sclerostin is also involved in inducing increased receptor activator of nuclear factor-k B ligand (RANKL) and decreased osteoprotegerin (OPG) expression in osteoblasts, contributing to the enhanced osteoclast activity occurring in patients with multiple myeloma-bone disease. Our data suggest that myeloma cells contribute to the suppression of bone formation through sclerostin secretion. Disclosures: No relevant conflicts of interest to declare.

The effects of protein kinase C (PKC) in the control of osteoclast activity are still unknown. We investigated the role of the enzyme in the control of microfilament organization, podosome assembly, bone resorption, and extracellular Ca2+ sensing in chicken and rabbit osteoclasts treated with agents known to affect PKC activity. Cells were treated for 20 min with a PKC activator [phorbol 12-myristate 13-acetate (PMA)], a PKC inhibitor (staurosporine), a protein kinase A (PKA) inhibitor (H-9), a guanosine 3',5'-cyclic monophosphate-dependent protein kinase-PKA-PKC inhibitor (H-7), or with the inactive phorbol, 4 alpha-phorbol, to examine microfilaments by decoration with rhodamine-phalloidin. In PMA-treated osteoclasts, the number of microfilament-containing adhesion structures (podosomes) per cell decreased. However, enlarged microfilamentous cores in podosomes and stress fiber-like filaments, otherwise absent in controls, appeared. Whereas H-7 induced increase of the number of podosomes, staurosporine, H-9, and 4 alpha-phorbol failed to change microfilament organization. Chicken osteoclasts received also long-term treatment with the agents in the presence of [3H]proline-prelabeled chicken or rat bone particles to measure bone resorption. PMA, as well as staurosporine and H-7, stimulated the resorbing activity, whereas cells were insensitive to H-9 and 4 alpha-phorbol. Measurement of cytosolic free calcium concentration in PMA-treated fura-2-loaded single osteoclasts demonstrated a synergistic effect of PKC activation on the inhibitory extracellular calcium concentration-sensing mechanism, which was, by contrast, blocked by H-7, staurosporine, and H-9 and was insensitive to 4 alpha-phorbol. These results indicate that PKC regulates osteoclast activity inducing both morphological and functional modifications.

Osteoblast-osteoclast relationships in bone resorption are unclear. We investigated whether osteoblasts constitutively influence osteoclast activity. We employed a serum-free co-culture system in which chicken osteoclasts and chick calvaria or, alternatively, isolated chick osteoblasts were cultured in two different compartments separated by a 0.45 micron porous membrane permeable to soluble molecules. Osteoclastic bone resorption, evaluated by release of 3H-proline from prelabeled bone fragments, was significantly enhanced by bone cells resident in the calvaria, as well as by isolated osteoblasts. Stimulation was specific, since periosteal cells, or skin fibroblasts, failed to mimic osteoblast activity. Conditioned medium from osteoblast cultures stimulated osteoclast function in a similar manner, indicating that paracrine signals, capable of crossing the porous membrane separating the two compartments, are released by the bone forming cells.

The effects of dental materials, intended for bone substitution, on cell growth and alkaline phosphatase activity of newborn rat periosteal cells have been studied in vitro. Confluent periosteal cells were exposed to three apatite-based materials (400 μg/mL) with different physico-chemical properties. The materials were a β-tricalcium phosphate with a microporous granular structure obtained by sinterization (Synthograft, Johnson & Johnson, East Windsor, NY), a 40-60-mesh microporous durapatite ceramic (Periograf, Sterling Drug, Inc., Rensselaer, NY), and a 1-2-mm-diameter hydroxyapatite ceramic (Osprovit, Feldmuhle Aktiengeselschaft, Plochingen, Germany) with macropores larger than 100 μm. Cell proliferation and alkaline phosphatase activity were assessed by incorporation of 3H-thymidine into trichloroacetic-acid-precipitable material and by a fluorimetric method, respectively. Cell viability and compatibility with the materials were determined by morphology in phase-contrast microscopy. Periosteal cells showed increased proliferation following exposure to Synthograft, but were unaffected by Osprovit, whereas Periograf caused significantly reduced cell growth. Alkaline phosphatase activity was unaffected by Osprovit, but was decreased by both Synthograft and Periograf. The results indicated a differential response of periosteal cells to bone-substituting materials with heterogeneous physico-chemical characteristics.

Although osteoclasts (OCs) differentiate under the control of RANK/RANKL/OPG system, a number of inflammatory cytokines can contribute to increase osteoclastogenesis in diseases associated with bone loss. Recently, different studies indicate that TRAIL is implicated in modulating osteoclastogenesis. Here, we investigated the effect of TRAIL on OC formation in physiological and pathological conditions with bone involvement utilizing osteoclastogenesis in vitro models represented by peripheral blood mononuclear cells (PBMCs) from healthy donors and patients affected by multiple myeloma or periodontal disease. We demonstrated that in PBMCs from healthy donors TRAIL can directly induce OC formation in the absence of RANKL, while exert an inhibitory effect when added concomitantly to RANKL. In PBMCs from the patients, in which media the levels of TRAIL, RANKL and OPG are elevated, the neutralization of TRAIL partially inhibits the OC formation, and this effect was reversed by RANKL addition. Finally, we detect high TRAIL levels in the sera from the patients. In conclusion, our results indicate that TRAIL could exert a different role in modulating OC differentiation in physiological and pathological conditions.

In this study the expression of voltage-dependent calcium channels on osteoclast plasma membrane has been investigated. We found that osteoclasts were sensitive to KCl-induced depolarization. In this circumstance a 4 fold transient cytosolic calcium concentration ([Ca2+]i) increase was observed. This increase was dose-dependent. Its half maximal effect was achieved at 30 mM KCl. Voltage sensitive calcium channels in osteoclasts were inhibited by specific antagonists. Nicardipine, a dihydropyridine derivative, was the most effective, inducing complete block of the channels at 10(-6) M. Verapamil (phenylalkylamine) and diltiazem (benzodiazepine) were less effective. These results are consistent with the presence, on the osteoclast membrane, of L-type voltage-sensitive calcium channels.

Periodontal disease (Pd) is characterized by an increased osteoclast resorption and a decreased osteoblast (OB) bone formation. OBs obtained from alveolar bone of Periodontitis patients (Pp) undergo apoptosis in the presence of TNF-related apoptosis-inducing ligand (TRAIL). We studied the intracellular apoptotic pathway induced by TRAIL; TRAIL death (DR4, DR5) and decoy (DcR1, DcR2) receptors expression in Periodontitis patients' OBs (PpOBs), and we measured the concentration of TRAIL in the serum of Pp. We demonstrated that DNA fragmentation and activation of caspase-8 and caspase-3 in PpOBs, following TRAIL stimulation, occurred in shorter time; moreover, a higher amount of both caspases was activated in order to direct OBs. Down-regulation of DcR2 in PpOBs was demonstrated and high TRAIL levels were detected in the serum of Pp. In conclusion, our data suggest that PpOBs are more sensitive to TRAIL-induced apoptosis when compared to the control group. The down-regulation of DcR2 possibly leads to an imbalanced ratio between death and decoy receptors. Our findings highlight a role of TRAIL in the pathogenesis of Pd.