Publications by authors named "Alberta Zallone"

61 Publications

Oxytocin regulates body composition.

Proc Natl Acad Sci U S A 2019 Dec 16. Epub 2019 Dec 16.

The Mount Sinai Bone Program, Icahn School of Medicine at Mount Sinai, New York, NY 10029;

The primitive neurohypophyseal nonapeptide oxytocin (OXT) has established functions in parturition, lactation, appetite, and social behavior. We have shown that OXT has direct actions on the mammalian skeleton, stimulating bone formation by osteoblasts and modulating the genesis and function of bone-resorbing osteoclasts. We deleted OXT receptors (OXTRs) selectively in osteoblasts and osteoclasts using and mice, respectively. Both male and female : mice recapitulate the low-bone mass phenotype of mice, suggesting that OXT has a prominent osteoblastic action in vivo. Furthermore, abolishment of the anabolic effect of estrogen in : mice suggests that osteoblastic OXTRs are necessary for estrogen action. In addition, the high bone mass in : mice indicates a prominent action of OXT in stimulating osteoclastogenesis. In contrast, we found that in pregnant and lactating : mice, elevated OXT inhibits bone resorption and rescues the bone loss otherwise noted during pregnancy and lactation. However, OXT does not contribute to ovariectomy-induced bone loss. Finally, we show that OXT acts directly on OXTRs on adipocytes to suppress the white-to-beige transition gene program. Despite this direct antibeiging action, injected OXT reduces total body fat, likely through an action on OXT-ergic neurons. Consistent with an antiobesity action of OXT, and mice display increased total body fat. Overall, the actions of OXT on bone mass and body composition provide the framework for future therapies for osteoporosis and obesity.
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http://dx.doi.org/10.1073/pnas.1913611116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936484PMC
December 2019

Isolation and Generation of Osteoclasts.

Methods Mol Biol 2019 ;1914:3-19

Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, L'Aquila, Italy.

This chapter describes the isolation, culture, and staining of osteoclasts. The key advantages of this assay are that it allows direct measurement of osteoclast number, bone resorption, as well as yielding good quantities of osteoclasts at defined stages of formation for molecular analysis. An additional focus of this chapter will be the generation of osteoclasts from less conventional animal species and cell lines.
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http://dx.doi.org/10.1007/978-1-4939-8997-3_1DOI Listing
June 2019

Actions of pituitary hormones beyond traditional targets.

J Endocrinol 2018 06 19;237(3):R83-R98. Epub 2018 Mar 19.

The Mount Sinai Bone Program, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

Studies over the past decade have challenged the long-held belief that pituitary hormones have singular functions in regulating specific target tissues, including master hormone secretion. Our discovery of the action of thyroid-stimulating hormone (TSH) on bone provided the first glimpse into the non-traditional functions of pituitary hormones. Here we discuss evolving experimental and clinical evidence that growth hormone (GH), follicle-stimulating hormone (FSH), adrenocorticotrophic hormone (ACTH), prolactin, oxytocin and arginine vasopressin (AVP) regulate bone and other target tissues, such as fat. Notably, genetic and pharmacologic FSH suppression increases bone mass and reduces body fat, laying the framework for targeting the FSH axis for treating obesity and osteoporosis simultaneously with a single agent. Certain 'pituitary' hormones, such as TSH and oxytocin, are also expressed in bone cells, providing local paracrine and autocrine networks for the regulation of bone mass. Overall, the continuing identification of new roles for pituitary hormones in biology provides an entirely new layer of physiologic circuitry, while unmasking new therapeutic targets.
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http://dx.doi.org/10.1530/JOE-17-0680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924585PMC
June 2018

The V2 receptor antagonist tolvaptan raises cytosolic calcium and prevents AQP2 trafficking and function: an in vitro and in vivo assessment.

J Cell Mol Med 2017 09 21;21(9):1767-1780. Epub 2017 Mar 21.

Department of Biosciences Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, Bari, Italy.

Tolvaptan, a selective vasopressin V2 receptor antagonist, is a new generation diuretic. Its clinical efficacy is in principle due to impaired vasopressin-regulated water reabsorption via aquaporin-2 (AQP2). Nevertheless, no direct in vitro evidence that tolvaptan prevents AQP2-mediated water transport, nor that this pathway is targeted in vivo in patients with syndrome of inappropriate antidiuresis (SIAD) has been provided. The effects of tolvaptan on the vasopressin-cAMP/PKA signalling cascade were investigated in MDCK cells expressing endogenous V2R and in mouse kidney. In MDCK, tolvaptan prevented dDAVP-induced increase in ser256-AQP2 and osmotic water permeability. A similar effect on ser256-AQP2 was found in V1aR -/- mice, thus confirming the V2R selectively. Of note, calcium calibration in MDCK showed that tolvaptan per se caused calcium mobilization from the endoplasmic reticulum resulting in a significant increase in basal intracellular calcium. This effect was only observed in cells expressing the V2R, indicating that it requires the tolvaptan-V2R interaction. Consistent with this finding, tolvaptan partially reduced the increase in ser256-AQP2 and the water permeability in response to forskolin, a direct activator of adenylyl cyclase (AC), suggesting that the increase in intracellular calcium is associated with an inhibition of the calcium-inhibitable AC type VI. Furthermore, tolvaptan treatment reduced AQP2 excretion in two SIAD patients and normalized plasma sodium concentration. These data represent the first detailed demonstration of the central role of AQP2 blockade in the aquaretic effect of tolvaptan and underscore a novel effect in raising intracellular calcium that can be of significant clinical relevance.
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http://dx.doi.org/10.1111/jcmm.13098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5571526PMC
September 2017

Pituitary-bone connection in skeletal regulation.

Horm Mol Biol Clin Investig 2016 Nov;28(2):85-94

Pituitary hormones have traditionally been thought to exert specific, but limited function on target tissues. More recently, the discovery of these hormones and their receptors in organs such as the skeleton suggests that pituitary hormones have more ubiquitous functions. Here, we discuss the interaction of growth hormone (GH), follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), adrenocorticotrophic hormone (ACTH), prolactin, oxytocin and arginine vasopressin (AVP) with bone. The direct skeletal action of pituitary hormones therefore provides new insights and therapeutic opportunities for metabolic bone diseases, prominently osteoporosis.
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http://dx.doi.org/10.1515/hmbci-2016-0015DOI Listing
November 2016

Functions of vasopressin and oxytocin in bone mass regulation.

Proc Natl Acad Sci U S A 2016 Jan 22;113(1):164-9. Epub 2015 Dec 22.

The Mount Sinai Bone Program, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029;

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.
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http://dx.doi.org/10.1073/pnas.1523762113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711832PMC
January 2016

The "love hormone" oxytocin regulates the loss and gain of the fat-bone relationship.

Front Endocrinol (Lausanne) 2015 18;6:79. Epub 2015 May 18.

Department of Basic Medical Science, Neuroscience and Sense Organs, University of Bari , Bari , Italy.

The involvement of oxytocin (OT) in bone metabolism is an interesting area of research that recently achieved remarkable results. Moreover, several lines of evidence have largely demonstrated that OT also participates in the regulation of energy metabolism. Hence, it has recently been determined that the posterior pituitary hormone OT directly regulates bone mass: mice lacking OT or OT receptor display severe osteopenia, caused by impaired bone formation. OT administration normalizes ovariectomy-induced osteopenia, bone marrow adiposity, body weight, and intra-abdominal fat depots in mice. This effect is mediated through inhibition of adipocyte precursor differentiation and reduction of adipocyte size. The exquisite role of OT in regulating the bone-fat connection adds another milestone to the biological evidence supporting the existence of a tight relationship between the adipose tissue and the skeleton.
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http://dx.doi.org/10.3389/fendo.2015.00079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4435037PMC
June 2015

In memoriam: the life and works of Itai Bab (1945-2014).

J Bone Miner Res 2015 Jan;30(1):1-2

Mount Sinai Bone Program, Icahn School of Medicine at Mount Sinai,, New York, NY, USA.

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http://dx.doi.org/10.1002/jbmr.2402DOI Listing
January 2015

Bisphosphonates inactivate human EGFRs to exert antitumor actions.

Proc Natl Acad Sci U S A 2014 Dec 1;111(50):17989-94. Epub 2014 Dec 1.

Departments of Medicine, Pediatrics, and Chemical and Structural Biology, and the Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029;

Bisphosphonates are the most commonly prescribed medicines for osteoporosis and skeletal metastases. The drugs have also been shown to reduce cancer progression, but only in certain patient subgroups, suggesting that there is a molecular entity that mediates bisphosphonate action on tumor cells. Using connectivity mapping, we identified human epidermal growth factor receptors (human EGFR or HER) as a potential new molecular entity for bisphosphonate action. Protein thermal shift and cell-free kinase assays, together with computational modeling, demonstrated that N-containing bisphosphonates directly bind to the kinase domain of HER1/2 to cause a global reduction in downstream signaling. By doing so, the drugs kill lung, breast, and colon cancer cells that are driven by activating mutations or overexpression of HER1. Knocking down HER isoforms thus abrogates cell killing by bisphosphonates, establishing complete HER dependence and ruling out a significant role for other receptor tyrosine kinases or the enzyme farnesyl pyrophosphate synthase. Consistent with this finding, colon cancer cells expressing low levels of HER do not respond to bisphosphonates. The results suggest that bisphosphonates can potentially be repurposed for the prevention and therapy of HER family-driven cancers.
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http://dx.doi.org/10.1073/pnas.1421410111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4273397PMC
December 2014

Repurposing of bisphosphonates for the prevention and therapy of nonsmall cell lung and breast cancer.

Proc Natl Acad Sci U S A 2014 Dec 1;111(50):17995-8000. Epub 2014 Dec 1.

Departments of Medicine, Pediatrics, and Chemical and Structural Biology, and the Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029;

A variety of human cancers, including nonsmall cell lung (NSCLC), breast, and colon cancers, are driven by the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases. Having shown that bisphosphonates, a class of drugs used widely for the therapy of osteoporosis and metastatic bone disease, reduce cancer cell viability by targeting HER1, we explored their potential utility in the prevention and therapy of HER-driven cancers. We show that bisphosphonates inhibit colony formation by HER1(ΔE746-A750)-driven HCC827 NSCLCs and HER1(wt)-expressing MB231 triple negative breast cancers, but not by HER(low)-SW620 colon cancers. In parallel, oral gavage with bisphosphonates of mice xenografted with HCC827 or MB231 cells led to a significant reduction in tumor volume in both treatment and prevention protocols. This result was not seen with mice harboring HER(low) SW620 xenografts. We next explored whether bisphosphonates can serve as adjunctive therapies to tyrosine kinase inhibitors (TKIs), namely gefitinib and erlotinib, and whether the drugs can target TKI-resistant NSCLCs. In silico docking, together with molecular dynamics and anisotropic network modeling, showed that bisphosphonates bind to TKIs within the HER1 kinase domain. As predicted from this combinatorial binding, bisphosphonates enhanced the effects of TKIs in reducing cell viability and driving tumor regression in mice. Impressively, the drugs also overcame erlotinib resistance acquired through the gatekeeper mutation T790M, thus offering an option for TKI-resistant NSCLCs. We suggest that bisphosphonates can potentially be repurposed for the prevention and adjunctive therapy of HER1-driven cancers.
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http://dx.doi.org/10.1073/pnas.1421422111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4273392PMC
December 2014

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

Proc Natl Acad Sci U S A 2014 Nov 5;111(46):16502-7. Epub 2014 Nov 5.

Mount Sinai Bone Program and Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029; Structural and Chemical Biology, Mount Sinai School of Medicine, New York, NY 10029

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.
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http://dx.doi.org/10.1073/pnas.1419349111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246276PMC
November 2014

Oxytocin and bone.

Am J Physiol Regul Integr Comp Physiol 2014 Oct 10;307(8):R970-7. Epub 2014 Sep 10.

Department of Basic Medical Science, Neuroscience and Sense Organs, University of Bari, Bari, Italy; and.

One of the most meaningful results recently achieved in bone research has been to reveal that the pituitary hormones have profound effect on bone, so that the pituitary-bone axis has become one of the major topics in skeletal physiology. Here, we discuss the relevant evidence about the posterior pituitary hormone oxytocin (OT), previously thought to exclusively regulate parturition and breastfeeding, which has recently been established to directly regulate bone mass. Both osteoblasts and osteoclasts express OT receptors (OTR), whose stimulation enhances bone mass. Consistent with this, mice deficient in OT or OTR display profoundly impaired bone formation. In contrast, bone resorption remains unaffected in OT deficiency because, even while OT stimulates the genesis of osteoclasts, it inhibits their resorptive function. Furthermore, in addition to its origin from the pituitary, OT is also produced by bone marrow osteoblasts acting as paracrine-autocrine regulator of bone formation modulated by estrogens. In turn, the power of estrogen to increase bone mass is OTR-dependent. Therefore, OTR(-/-) mice injected with 17β-estradiol do not show any effects on bone formation parameters, while the same treatment increases bone mass in wild-type mice. These findings together provide evidence for an anabolic action of OT in regulating bone mass and suggest that bone marrow OT may enhance the bone-forming action of estrogen through an autocrine circuit. This established new physiological role for OT in the maintenance of skeletal integrity further suggests the potential use of this hormone for the treatment of osteoporosis.
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http://dx.doi.org/10.1152/ajpregu.00040.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200383PMC
October 2014

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

Proc Natl Acad Sci U S A 2013 Nov 28;110(46):18644-9. Epub 2013 Oct 28.

Department of Histology, University of Bari, 70124 Bari, Italy.

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.
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http://dx.doi.org/10.1073/pnas.1318257110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831977PMC
November 2013

Aortic valvular interstitial cells apoptosis and calcification are mediated by TNF-related apoptosis-inducing ligand.

Int J Cardiol 2013 Nov 5;169(4):296-304. Epub 2013 Oct 5.

Department of Emergencies and Organ Transplantation (DETO), Division of Cardiac Surgery, University "Aldo Moro" of Bari, Italy; Department of Basic Medical Sciences, Neurosciences and Sense Organs, Section of Human Anatomy and Histology, University "Aldo Moro" of Bari, Italy.

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.
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http://dx.doi.org/10.1016/j.ijcard.2013.09.012DOI Listing
November 2013

Genetic confirmation for a central role for TNFα in the direct action of thyroid stimulating hormone on the skeleton.

Proc Natl Acad Sci U S A 2013 Jun 28;110(24):9891-6. Epub 2013 May 28.

The Mount Sinai Bone Program and Department of Medicine and Pediatrics, Mount Sinai School of Medicine, New York, NY 10029, USA.

Clinical data showing correlations between low thyroid-stimulating hormone (TSH) levels and high bone turnover markers, low bone mineral density, and an increased risk of osteoporosis-related fractures are buttressed by mouse genetic and pharmacological studies identifying a direct action of TSH on the skeleton. Here we show that the skeletal actions of TSH deficiency are mediated, in part, through TNFα. Compound mouse mutants generated by genetically deleting the Tnfα gene on a Tshr(-/-) (homozygote) or Tshr(+/-) (heterozygote) background resulted in full rescue of the osteoporosis, low bone formation, and hyperresorption that accompany TSH deficiency. Studies using ex vivo bone marrow cell cultures showed that TSH inhibits and stimulates TNFα production from macrophages and osteoblasts, respectively. TNFα, in turn, stimulates osteoclastogenesis but also enhances the production in bone marrow of a variant TSHβ. This locally produced TSH suppresses osteoclast formation in a negative feedback loop. We speculate that TNFα elevations due to low TSH signaling in human hyperthyroidism contribute to the bone loss that has traditionally been attributed solely to high thyroid hormone levels.
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http://dx.doi.org/10.1073/pnas.1308336110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683712PMC
June 2013

Osteoblasts display different responsiveness to TRAIL-induced apoptosis during their differentiation process.

Cell Biochem Biophys 2013 ;67(3):1127-36

Section of Human Anatomy and Histology - R. Amprino, Department of Basic Medical Sciences, Neuroscience and Sense Organs, Medical School, University of Bari, Piazza Giulio Cesare 11, 70124, Bari, Italy.

Apoptosis can occur throughout the life span of osteoblasts (OBs), beginning from the early stages of differentiation and continuing throughout all stages of their working life. Here, we investigated the effects of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal human OBs showing for the first time that the expression of TRAIL receptors is modulated during OB differentiation. In particular, the TRAIL receptor ratio was in favor of the deaths because of the low expression of DcR2 in undifferentiated OBs, differently it was shifted toward the decoys in differentiated ones. Undifferentiated OBs treated with TRAIL showed reduced cell viability, whereas differentiated OBs displayed TRAIL resistance. The OB sensitiveness to TRAIL was due to the up-regulation of DR5 and the down-regulation of DcR2. The main death receptor involved in TRAIL-reduced OB viability was DR5 as demonstrated by the rescue of cell viability observed in the presence of anti-DR5 neutralizing antibody. Besides the ratio of TRAIL receptors, the sensitivity of undifferentiated OBs to TRAIL-cytotoxic effect was also associated with low mRNA levels of intracellular anti-apoptotic proteins, such as cFLIP, the activation of caspase-8 and -3, as well as the DNA fragmentation. This study suggests that apoptotic effect exerted by TRAIL/TRAIL-receptor system on normal human OB is strictly dependent upon cell differentiation status.
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http://dx.doi.org/10.1007/s12013-013-9616-6DOI Listing
July 2014

Activation of the receptor activator of the nuclear factor-κB ligand pathway during coronary bypass surgery: comparison between on- and off-pump coronary artery bypass surgery procedures.

Eur J Cardiothorac Surg 2013 Aug 12;44(2):e141-7. Epub 2013 May 12.

Department of Emergencies and Organ Transplantation, Division of Cardiac Surgery, University Aldo Moro of Bari, Bari, Italy.

Objectives: The receptor activator of the nuclear factor kappa-B (NF-κB) ligand (RANKL), its membrane receptor RANK and its decoy receptor osteoprotegerin (OPG) are all members of the tumour necrosis factor family involved in bone metabolism and immune response. We evaluated the activation of the OPG/RANKL/RANK pathway in patients undergoing cardiac surgery with and without cardiopulmonary bypass (CPB).

Methods: Twenty consecutive patients undergoing elective coronary artery surgery were enrolled in the study and assigned either to the on-pump or to the off-pump group. Pre- and postoperative serum levels of OPG and RANKL were evaluated by enzyme-linked immunosorbent assay; gene expression of OPG, RANKL, RANK and NF-κB p50 subunits were determined by real-time polymerase chain reaction in peripheral blood T-cells and monocytes.

Results: Serum levels of OPG significantly increased after surgery in both groups, whereas serum levels of RANKL did not differ over time. T-cells from the on-pump group showed increased gene expression of OPG, RANKL and RANK after the intervention, whereas no mRNA variation for these genes was detected in T-cells from off-pump patients. Gene expression of p50 subunit increased in T-cells and monocytes from both groups.

Conclusions: Cardiac surgery induces the activation of the OPG/RANKL/RANK pathway; both on- and off-pump procedures are associated with increased postoperative OPG serum levels and up-regulation of the NF-κB p50 subunit.
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http://dx.doi.org/10.1093/ejcts/ezt244DOI Listing
August 2013

Skeletal parasympathetic innervation communicates central IL-1 signals regulating bone mass accrual.

Proc Natl Acad Sci U S A 2012 Sep 4;109(38):15455-60. Epub 2012 Sep 4.

Bone Laboratory, Hebrew University of Jerusalem, Jerusalem 91120, Israel.

Bone mass accrual is a major determinant of skeletal mass, governed by bone remodeling, which consists of bone resorption by osteoclasts and bone formation by osteoblasts. Bone mass accrual is inhibited by sympathetic signaling centrally regulated through activation of receptors for serotonin, leptin, and ACh. However, skeletal activity of the parasympathetic nervous system (PSNS) has not been reported at the bone level. Here we report skeletal immune-positive fibers for the PSNS marker vesicular ACh transporter (VAChT). Pseudorabies virus inoculated into the distal femoral metaphysis is identifiable in the sacral intermediolateral cell column and central autonomic nucleus, demonstrating PSNS femoral innervation originating in the spinal cord. The PSNS neurotransmitter ACh targets nicotinic (nAChRs), but not muscarinic receptors in bone cells, affecting mainly osteoclasts. nAChR agonists up-regulate osteoclast apoptosis and restrain bone resorption. Mice deficient of the α(2)nAChR subunit have increased bone resorption and low bone mass. Silencing of the IL-1 receptor signaling in the central nervous system by brain-specific overexpression of the human IL-1 receptor antagonist (hIL1ra(Ast)(+/+) mice) leads to very low skeletal VAChT expression and ACh levels. These mice also exhibit increased bone resorption and low bone mass. In WT but not in hIL1ra(Ast)(+/+) mice, the cholinergic ACh esterase inhibitor pyridostigmine increases ACh levels and bone mass apparently by inhibiting bone resorption. Taken together, these results identify a previously unexplored key central IL-1-parasympathetic-bone axis that antagonizes the skeletal sympathetic tone, thus potently favoring bone mass accrual.
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http://dx.doi.org/10.1073/pnas.1206061109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458367PMC
September 2012

Blocking antibody to the β-subunit of FSH prevents bone loss by inhibiting bone resorption and stimulating bone synthesis.

Proc Natl Acad Sci U S A 2012 Sep 20;109(36):14574-9. Epub 2012 Aug 20.

Mount Sinai Bone Program, Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.

Low estrogen levels undoubtedly underlie menopausal bone thinning. However, rapid and profuse bone loss begins 3 y before the last menstrual period, when serum estrogen is relatively normal. We have shown that the pituitary hormone FSH, the levels of which are high during late perimenopause, directly stimulates bone resorption by osteoclasts. Here, we generated and characterized a polyclonal antibody to a 13-amino-acid-long peptide sequence within the receptor-binding domain of the FSH β-subunit. We show that the FSH antibody binds FSH specifically and blocks its action on osteoclast formation in vitro. When injected into ovariectomized mice, the FSH antibody attenuates bone loss significantly not only by inhibiting bone resorption, but also by stimulating bone formation, a yet uncharacterized action of FSH that we report herein. Mesenchymal cells isolated from mice treated with the FSH antibody show greater osteoblast precursor colony counts, similarly to mesenchymal cells isolated from FSH receptor (FSHR)(-/-) mice. This suggests that FSH negatively regulates osteoblast number. We confirm that this action is mediated by signaling-efficient FSHRs present on mesenchymal stem cells. Overall, the data prompt the future development of an FSH-blocking agent as a means of uncoupling bone formation and bone resorption to a therapeutic advantage in humans.
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http://dx.doi.org/10.1073/pnas.1212806109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437842PMC
September 2012

Effects of radial shock waves therapy on osteoblasts activities.

Musculoskelet Surg 2012 Dec 27;96(3):183-9. Epub 2012 Jul 27.

Department of Neuroscience and Organs of Sense, Orthopedics Section, Faculty of Medicine and Surgery, General Hospital, University of Bari, Piazza Giulio Cesare 11, 70124 Bari, Italy.

Radial shock waves therapy (RSWT) differs from extracorporeal shock waves therapy (ESWT) in that it produces a non-focused wave that is dissipated radially at the skin. Few studies have yet explored the effects of RSWT on bone tissue. Osteoblasts in culture flasks were studied by polymerase chain reaction after treatment with RSW (500 impulses, 0.05 mJ/mm(2)). An inhibited osteoblastogenesis was observed, with a statistically significant reduction in type 1 collagen, osterix, bone sialoprotein and receptor activator NF kappa ligand expression at 24 and 48 h, of osteocalcin at 24, 48 and 72 h, and osteopontin at 48 and 72 h. These findings show that RSWT is not indicated for treatment of delayed fracture union, pseudoarthrosis, and complex regional pain syndrome. The observed reduction in the receptor activator of nuclear factor-kB ligand/osteoprotegerin ratio suggests that it has an inhibiting effect on osteoclastogenesis, which could make it a useful tool for applications in proliferative diseases.
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http://dx.doi.org/10.1007/s12306-012-0213-4DOI Listing
December 2012

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

J Biol Chem 2012 Aug 2;287(34):29159-67. Epub 2012 Jul 2.

Mount Sinai Bone Program, Mount Sinai School of Medicine, New York 10029, USA.

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(+)/OTR(fl/fl) mice, but not TRAP-Cre(+)/OTR(fl/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.
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http://dx.doi.org/10.1074/jbc.M112.365049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436530PMC
August 2012

Osteoblast and osteoclast crosstalks: from OAF to Ephrin.

Inflamm Allergy Drug Targets 2012 Jun;11(3):196-200

Department of Basic Medical Sciences, Policlinico, Piazza G. Cesare 11, 70124 Bari, Italy.

The maintenance of bone homeostasis is tightly controlled, and largely dependent upon cellular communication between osteoclasts and osteoblasts, and the coupling of bone resorption to bone formation. This tight coupling is essential for the correct function and maintenance of the skeletal system, repairing microscopic skeletal damage and replacing aged bone. Cells in osteoclast and osteoblast lineages communicate with each other through diffusible paracrine factors, cell-cell contact, and cell-bone matrix interaction. Osteoclast-osteoblast communication occurs in a basic multicellular unit (BMU) at the initiation, transition and termination phases of bone remodeling. At the initiation phase, hematopoietic precursors are recruited to the BMU. These precursors differentiate into osteoclasts following interactions with osteoblasts, which express and/or secrete ligands as RANK-L and OPG. Subsequently, the transition from bone resorption to formation is mediated by osteoclast-derived 'coupling factors', which direct the differentiation and activation of osteoblasts in resorbed lacunae to refill it with new bone. Signals derived from molecules released from the resorbed bone matrix, as TGF-beta and bidirectional signaling generated by interaction between ephrinB2 on osteoclasts and EphB4 on osteoblast precursors facilitates the transition. At the termination phase, bone remodeling is completed by osteoblastic bone formation and mineralization of bone matrix. The research steps that brought to the present knowledge are summarized in this review.
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http://dx.doi.org/10.2174/187152812800392670DOI Listing
June 2012

Adrenergic stimulation decreases osteoblast oxytocin synthesis.

Ann N Y Acad Sci 2011 Nov;1237:53-7

Department of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy.

Oxytocin (OT) regulates bone mass by inducing the differentiation of osteoblasts to a mature, mineralizing phenotype. We have shown recently that osteoblasts can synthesize OT. In view of known interactions between OT-ergic and adrenergic neurons in the central nervous system, we questioned whether the negative regulation of osteoblast differentiation by adrenergic nerves was mediated through its suppression of OT synthesis. We first confirmed that α(1b) and β(2) adrenergic receptors were expressed on both primary murine osteoblasts and MC3T3-E1 cells. We then showed that α(1) and β(2) adrenergic agonists downregulated OT synthesis, and that the effect of each agonist was reversed by its respective antagonist. The data suggest that the negative effects of adrenergic stimulation on bone mass could, in part, arise from decreased OT synthesis.
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http://dx.doi.org/10.1111/j.1749-6632.2011.06235.xDOI Listing
November 2011

Sclerostin is overexpressed by plasma cells from multiple myeloma patients.

Ann N Y Acad Sci 2011 Nov;1237:19-23

Department of Basic Medical Science, Section of Human Anatomy and Histology, University of Bari, Bari, Italy.

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.
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http://dx.doi.org/10.1111/j.1749-6632.2011.06196.xDOI Listing
November 2011

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

Biochem Biophys Res Commun 2011 Aug 29;411(3):512-5. Epub 2011 Jun 29.

Department of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy.

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.
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http://dx.doi.org/10.1016/j.bbrc.2011.06.158DOI Listing
August 2011

A 3D in vitro bone organ model using human progenitor cells.

Eur Cell Mater 2011 May 15;21:445-58; discussion 458. Epub 2011 May 15.

Department of Surgery, University Hospital Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland.

Three-dimensional (3D) organotypic culture models based on human cells may reduce the use of complex and costly animal models, while gaining clinical relevance. This study aimed at developing a 3D osteoblastic-osteoclastic-endothelial cell co-culture system, as an in vitro model to mimic the process of bone turnover. Osteoprogenitor and endothelial lineage cells were isolated from the stromal vascular fraction (SVF) of human adipose tissue, whereas CD14+ osteoclast progenitors were derived from human peripheral blood. Cells were co-cultured within 3D porous ceramic scaffolds using a perfusion-based bioreactor device, in the presence of typical osteoclastogenic factors. After 3 weeks, the scaffolds contained cells with endothelial (2.0±0.3%), pre/osteoclastic (14.0±1.4%) and mesenchymal/osteoblastic (44.0±8.4%) phenotypes, along with tartrate-resistant acid phosphatase-positive (TRAP+) osteoclastic cells in contact with deposited bone-like matrix. Supernatant analysis demonstrated sustained matrix deposition (by C-terminus procollagen-I propeptides), resorption (by N-terminus collagen-I telopeptides and phosphate levels) and osteoclastic activity (by TRAP-5b) only when SVF and CD14+ cells were co-cultured. Scanning electron microscopy and magnetic resonance imaging confirmed the pattern of matrix deposition and resorption. The effectiveness of Vitamin D in replacing osteoclastogenic factors indicated a functional osteoblast-osteoclast coupling in the system. The formation of human-origin bone-like tissue, blood vessels and osteoclasts upon ectopic implantation validated the functionality of the developed cell types. The 3D co-culture system and the associated non-invasive analytical tools can be used as an advanced model to capture some aspects of the functional coupling of bone-like matrix deposition and resorption and could be exploited toward the engineering of multi-functional bone substitute implants.
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http://dx.doi.org/10.22203/ecm.v021a33DOI Listing
May 2011

Effect of shock wave treatment on platelet-rich plasma added to osteoblast cultures.

Ultrasound Med Biol 2011 Jan 16;37(1):160-8. Epub 2010 Nov 16.

Department of Clinical Methodology and Surgical Techniques, Orthopedics Section, Faculty of Medicine and Surgery of University of Bari, General Hospital, Bari, Italy.

The aim of this study was to verify the effects on osteoblast cultures of adding a platelet-rich plasma (PRP) concentrate pretreated with 500 shock wave (SW) at an energy flow density of 0.17 mJ/mm(2), emitted by an electromagnetic generator Minilith SL1 (STORZ, Germany), reproducing the conditions of our previous study in which we apply SW directly on osteoblasts. Real-time PCR showed that in osteoblast cultures with added PRP pretreated with SW, there was an increased expression at 48 h of insulin-like growth factor binding protein 3 (IGFBP-3) and runt-related transcription factor 2 (RUNX2) and at 72 h, of collagen type I, osteocalcin, insulin-like growth factor 1 (IGF-1) as well as IGFBP-3. Western blotting confirmed the increased protein synthesis of IGFBP-3. This experience suggests that extracorporeal shock wave treatment (ESWT) should stimulate osteogenesis also by indirect platelets-mediated network. It therefore seems possible that combining the two methods, ESWT and bioengineering procedures to infiltrate PRP and growth factors, could be a successful approach.
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http://dx.doi.org/10.1016/j.ultrasmedbio.2010.10.016DOI Listing
January 2011

The formation of osteoclasts in multiple myeloma bone disease patients involves the secretion of soluble decoy receptor 3.

Ann N Y Acad Sci 2010 Mar;1192:298-302

Department of Human Anatomy and Histology, University of Bari, Bari, Italy.

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.
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http://dx.doi.org/10.1111/j.1749-6632.2009.05304.xDOI Listing
March 2010

Role of the pituitary-bone axis in skeletal pathophysiology.

Curr Opin Endocrinol Diabetes Obes 2009 Dec;16(6):423-9

The Mount Sinai Bone Program, Mount Sinai School of Medicine, New York, New York, USA.

Purpose Of Review: Embedded within textbooks for decades is the hard fact that releasing hormones from the anterior pituitary, namely, follicle-stimulating hormone, thyroid-stimulating hormone and adrenocorticotropic hormone, stimulate master hormone secretion from target endocrine organs. We propose a paradigm shift in endocrine physiology, which is that these hormones act by design on bone directly, also now considered an endocrine organ.

Recent Findings: Complementary investigations using mouse genetic and cell biological approaches reveal that follicle-stimulating hormone and thyroid-stimulating hormone act on bone cells directly to regulate bone remodeling and bone mass. Thyroid-stimulating hormone inhibits bone remodeling, whereas follicle-stimulating hormone stimulates it. We also find that the posterior pituitary hormone oxytocin is anabolic to the skeleton.

Summary: An ambitious extrapolation is that a plurality of pituitary hormones acts in concert as part of a 'pituitary-bone' axis to regulate skeletal integrity in health and disease. When dysregulated master hormone levels during hypogonadism and hyperthyroidism cause altered pituitary hormone secretion through hypothalamic feedback, the latter hormones contribute to the skeletal loss.
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http://dx.doi.org/10.1097/MED.0b013e3283328aeeDOI Listing
December 2009