Roberto Tamma

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.

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)

Regulation of bone remodeling by vasopressin explains the bone loss in hyponatremia

Significance In this study, we show that a primitive brain hormone, arginine vasopressin (AVP), which has hitherto been implicated in regulating water balance in mammals, has a function in skeletal homeostasis. Using genetically modified mice that are lacking one of the AVP receptors as well as pharmacologic inhibitors, we show that AVP negatively regulates osteoblasts (cells that form new bone) and stimulates osteoclasts (cells that remove old bone). Our findings explain the bone loss that is known to accompany low blood sodium levels in patients when AVP levels are high. 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.

Extracorporeal shock waves stimulate osteoblast activities.

The extracorporeal shock wave therapy (ESWT) is an extensively applied treatment for musculoskeletal disorders because it promotes bone repair. The aim of this study was to evaluate the direct effect of ESWT on murine osteoblasts to clarify the cellular mechanism that leads to the induction of osteogenesis. Osteoblasts in culture flasks were treated with ESWT pulses (500 impulses of 0.05 mJ/mm(2)) generated by an electromagnetic device. Using western blot analysis 3h after ESWT, an increased expression of Bax was found, indicating a fast pro-apoptotic effect of treatment on some of the osteoblasts. Activation of the cyclin E2/CDK2 is the complex that regulates the G1-S transition and is essential for cell proliferation. It was evident 24 to 72h after treatment, indicating a proliferative stimulus. A decreased expression of osteoprotegerin (OPG) and receptor activator NF kappa B ligand (RANKL) 24 and 48h after ESW, followed by a later increase of OPG, paired with a much smaller increase of RANKL, was evident by real-time polymerase chain reaction (PCR). The decreased RANKL/OPG ratio suggests inhibition of osteoclastogenesis. We can conclude that ESWT induces bone repair through the proliferation and differentiation of osteoblasts and the reduction of their secretion of pro-osteoclastogenic factors.

The death receptor DR5 is involved in TRAIL-mediated human osteoclast apoptosis

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.

Bone marrow oxytocin mediates the anabolic action of estrogen on the skeleton

Background: The mechanism underlying the anabolic effect of estrogen on the skeleton is unclear. Results: We report that estrogen-induced bone formation in mice occurs through oxytocin (OT) produced by osteoblasts in bone marrow. Conclusion: Feed-forward OT release in bone marrow by a rising estrogen level may facilitate rapid skeletal recovery after lactation. Significance: The study highlights a novel mechanism for estrogen action on bone. 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.

Soluble decoy receptor 3 modulates the survival and formation of osteoclasts from multiple myeloma bone disease patients

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 TNFα by T lymphocytes and only TNFα 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.

Regulated production of the pituitary hormone oxytocin from murine and human osteoblasts

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

Aquaporin-4 contributes to the resolution of peritumoural brain oedema in human glioblastoma multiforme after combined chemotherapy and radiotherapy

Brain tumour oedema is coupled with blood-brain barrier damage and alteration in water flow. Aquaporin-4 (AQP4) is involved in the development and resolution of brain oedema, and it is strongly upregulated in glioblastoma multiforme (GBM). Here, we evaluated AQP4 expression and content in GBM and correlated with VEGF-VEGFR-2 expression. In the relapse after chemotherapy and radiotherapy, AQP4 content reduced in parallel with VEGF-VEGFR-2, as compared with primary tumours, and in the peripheral areas of relapsed tumours AQP4 mimicked normal findings of perivascular rearrangement. After immunogold electron microscopy, gold particles were attached on the glial membrane facing the perivascular side, likewise AQP4 gold labelling of the vessels of the control areas. In primary tumours the peripheral vessels appeared faintly marked by AQP4, while the perivascular tumour cells showed a strong expression. The vasculature of the inner tumour areas was unlabelled by AQP4, while tumour cells were labelled, in both primary and relapsing tumours. Relapsed tumours after radiotherapy alone showed slight AQP4 reduction and perivascular restoring in the peripheral areas of the tumour. These data indicate that in GBM chemotherapy and radiotherapy induce a down-regulation in AQP4 expression restoring its perivascular rearrangement suggesting its potential role in the resolution of brain oedema.

Osteoblast regulation via ligand-activated nuclear trafficking of the oxytocin receptor

Significance We have shown previously that oxytocin (Oxt), other than regulating lactation and social bonding, is a potent stimulator of bone formation by the osteoblast. Here, we present evidence that this action is exerted through the nuclear localization of the Oxt receptor (Oxtr). Our findings prompt additional studies into the contribution of nuclear Oxtr signaling in regulating lactation and social bonding. 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.