Silvia Colucci

Oxytocin is an anabolic bone hormone

We report that oxytocin (OT), a primitive neurohypophyseal hormone, hitherto thought solely to modulate lactation and social bonding, is a direct regulator of bone mass. Deletion of OT or the OT receptor (Oxtr) in male or female mice causes osteoporosis resulting from reduced bone formation. Consistent with low bone formation, OT stimulates the differentiation of osteoblasts to a mineralizing phenotype by causing the up-regulation of BMP-2, which in turn controls Schnurri-2 and 3, Osterix, and ATF-4 expression. In contrast, OT has dual effects on the osteoclast. It stimulates osteoclast formation both directly, by activating NF-κB and MAP kinase signaling, and indirectly through the up-regulation of RANK-L. On the other hand, OT inhibits bone resorption by mature osteoclasts by triggering cytosolic Ca2+ release and NO synthesis. Together, the complementary genetic and pharmacologic approaches reveal OT as a novel anabolic regulator of bone mass, with potential implications for osteoporosis therapy.

Transforming Growth Factor-β1 Triggers Hepatocellular Carcinoma Invasiveness via α3β1 Integrin

Metastasis occurrence in the course of hepatocellular carcinoma (HCC) severely affects prognosis and survival. We have shown that HCC invasive cells express α3β1-integrin whereas noninvasive cells do not. Here we show that transforming growth factor (TGF)-β1 stimulates α3-integrin expression at a transcriptional level in noninvasive HCC cells, causing transformation into a motile and invasive phenotype. Such activities are inhibited by neutralizing anti-α3- but not anti-α6-integrin monoclonal antibodies. HCC invasive cells secrete abundant levels of active TGF-β1 in comparison with noninvasive cells, but in the latter, addition of active matrix metalloproteinases-2 increases the concentration of active TGF-β1. In this way, the cells express α3-integrin at a transcriptional level and acquire motility on Ln-5. By contrast, an anti-TGF-β1-neutralizing antibody reduces α3-integrin expression and the invasive ability of HCC invading cells. In HCC patients, TGF-β1 serum concentrations and α3-integrin expression are strongly correlated. The integrin, absent in normal and peritumoral liver parenchyma, is abundantly expressed in HCC primary and metastatic tissue. In particular, patients with metastasis show higher levels of TGF-β1 serum concentrations and stronger expression of TGF-β1 and α3-integrin in HCC tissues. In conclusion, TGF-β1 may play an important role in HCC invasiveness by stimulating α3-integrin expression, and could therefore be an important target for new therapies.

Synthesis, analytical characterization, and osteoblast adhesion properties on RGD-grafted polypyrrole coatings on titanium substrates

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.

Periodontal disease: linking the primary inflammation to bone loss.

Periodontal disease (PD), or periodontitis, is defined as a bacterially induced disease of the tooth-supporting (periodontal) tissues. It is characterized by inflammation and bone loss; therefore understanding how they are linked would help to address the most efficacious therapeutic approach. Bacterial infection is the primary etiology but is not sufficient to induce the disease initiation or progression. Indeed, bacteria-derived factors stimulate a local inflammatory reaction and activation of the innate immune system. The innate response involves the recognition of microbial components by host cells, and this event is mediated by toll-like receptors (TLRs) expressed by resident cells and leukocytes. Activation of these cells leads to the release of proinflammatory cytokines and recruitment of phagocytes and lymphocytes. Activation of T and B cells initiates the adaptive immunity with Th1 Th2 Th17 Treg response and antibodies production respectively. In this inflammatory scenario, cytokines involved in bone regulation and maintenance have considerable relevance because tissue destruction is believed to be the consequence of host inflammatory response to the bacterial challenge. In the present review, we summarize host factors including cell populations, cytokines, and mechanisms involved in the destruction of the supporting tissues of the tooth and discuss treatment perspectives based on this knowledge.

IL-7 Up-Regulates TNF-α-Dependent Osteoclastogenesis in Patients Affected by Solid Tumor

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-α 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.

Mechanisms of spontaneous osteoclastogenesis in cancer with bone involvement

Bone metastases represents a common cause of morbidity in patients suffering many types of cancer: breast, lung, kidney, prostate, and multiple myeloma. Osteolytic metastases often cause severe pain, pathologic fractures, hypercalcemia, spinal cord compression, and other nervecompression syndromes. Osteoclasts (OCs), cells deriving from granulocitic‐macrophagic lineage, are responsible for osteolysis, which may be reduced by inhibiting both OCs formation and activity. By studying bone osteolytic metastases mechanism in solid tumors, we report here our findings that cancer patients with bone involvement display an increase in osteoclasts precursors, compared with both healthy controls and cancer patients without bone metastases. Peripheral blood mononuclear cells (PBMCs) from patients with osteolytic lesions show osteoclastogenesis without adding M‐CSF, RANKL, or TNF‐α. However, these factors are necessary to generate OCs from healthy donors, non‐osteolytic patient PBMCs and T‐cell depleted PBMCs. OCs derived from cancer patients show more resorption pits than OCs from healthy donors and express genes involved in osteoclastogenesis. Our data show that a spontaneous osteoclastogenesis occurs in patients affected by osteolytic lesions and may be supported by factors released by T lymphocytes. These factors could give a priming to osteoclast precursors and promote osteoclastogenesis. In fact, T‐cell depleted PBMCs do not differentiate into OCs without adding M‐CSF and RANKL. Moreover, we do not obtain a higher number of OCs by increasing RANKL doses in cultures, and OCs and T lymphocytes mRNA level are detected for TNF‐α but not for RANKL. The addition of OPG to PBMCs cultures do not modify spontaneous osteoclastogenesis. A neutralizing anti‐TNF‐α antibody in unstimulated PBMC cultures of osteolytic cancer patients induces an inhibition of osteoclastogenesis. These data suggest that TNF‐α may be responsible for osteoclastogenesis in these tumors.

The myokine irisin increases cortical bone mass

Significance Although exercise is a well known and potent stimulus for new bone formation, and weightlessness or muscle loss characteristically cause bone loss, it has remained unclear how muscle talks to bone, despite their close proximity. Here, we show that a molecule irisin derived from skeletal muscle in response to exercise has profound effects in enhancing mass and improving the geometry and strength specifically of cortical bone, the key function of which is to resist bending and torsion. Trabecular bone, which is a reservoir for bodily calcium, is remarkably spared. Irisin may therefore not only be the molecule responsible for muscle–bone connectivity, but could also become a therapy for sarcopenia and osteoporosis, which occur in tandem in the elderly. It is unclear how physical activity stimulates new bone synthesis. We explored whether irisin, a newly discovered myokine released upon physical activity, displays anabolic actions on the skeleton. Young male mice were injected with vehicle or recombinant irisin (r-irisin) at a low cumulative weekly dose of 100 µg kg−1. We observed significant increases in cortical bone mass and strength, notably in cortical tissue mineral density, periosteal circumference, polar moment of inertia, and bending strength. This anabolic action was mediated primarily through the stimulation of bone formation, but with parallel notable reductions in osteoclast numbers. The trabecular compartment of the same bones was spared, as were vertebrae from the same mice. Higher irisin doses (3,500 µg kg−1 per week) cause browning of adipose tissue; this was not seen with low-dose r-irisin. Expectedly, low-dose r-irisin modulated the skeletal genes, Opn and Sost, but not Ucp1 or Pparγ expression in white adipose tissue. In bone marrow stromal cell cultures, r-irisin rapidly phosphorylated Erk, and up-regulated Atf4, Runx2, Osx, Lrp5, β-catenin, Alp, and Col1a1; this is consistent with a direct receptor-mediated action to stimulate osteogenesis. We also noted that, although the irisin precursor Fndc5 was expressed abundantly in skeletal muscle, other sites, such as bone and brain, also expressed Fndc5, albeit at low levels. Furthermore, muscle fibers from r-irisin–injected mice displayed enhanced Fndc5 positivity, and irisin induced Fdnc5 mRNA expression in cultured myoblasts. Our data therefore highlight a previously unknown action of the myokine irisin, which may be the molecular entity responsible for muscle–bone connectivity.

MYELOMA CELLS SUPPRESS OSTEOBLASTS THROUGH SCLEROSTIN SECRETION

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.

Lymphocytes and synovial fluid fibroblasts support osteoclastogenesis through RANKL, TNFα, and IL‐7 in an in vitro model derived from human psoriatic arthritis

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, TNFα, 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, TNFα, and IL‐7. Osteoclastogenesis was studied in the presence of RANK‐Fc, anti‐TNFα, 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 TNFα, while fibroblasts from synovial fluid produced only RANKL. We show that the presence of RANK‐Fc and/or anti‐TNFα 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, TNFα, and IL‐7, contributing to the aggressive bone resorption in PsA patients. Copyright

Microgravity during spaceflight directly affects in vitro osteoclastogenesis and bone resorption

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.— Tamma, R.,Colaianni, G., Camerino, C., Di Benedetto, A., Greco, G., Strippoli, M., Vergari, R., Grano, A., Mancini, L., Mori, G., Colucci, S., Grano, M., Zallone, A. Microgravity during spaceflight directly affects in vitro osteoclastogenesis and bone resorption. FASEB J. 23, 2549–2554 (2009)