Oxytocin regulates body composition.

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 Col2.3Cre and Acp5Cre mice, respectively. Both male and female Col2.3Cre+:Oxtrfl/fl mice recapitulate the low-bone mass phenotype of Oxtr+/- mice, suggesting that OXT has a prominent osteoblastic action in vivo. Furthermore, abolishment of the anabolic effect of estrogen in Col2.3Cre+:Oxtrfl/fl mice suggests that osteoblastic OXTRs are necessary for estrogen action. In addition, the high bone mass in Acp5Cre+:Oxtrfl/fl mice indicates a prominent action of OXT in stimulating osteoclastogenesis. In contrast, we found that in pregnant and lactating Col2.3Cre+:Oxtrfl/fl 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, Oxt-/- and Oxtr-/- 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.

Isolation and Generation of Osteoclasts.

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.

Actions of pituitary hormones beyond traditional targets.

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.

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

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.

Pituitary-bone connection in skeletal regulation.

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.

Functions of vasopressin and oxytocin in bone mass regulation.

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.

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

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.

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

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.

Bisphosphonates inactivate human EGFRs to exert antitumor actions.

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.

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

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 ß-arrestins (Arrbs), the small GTPase Rab5, importin-ß (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.

Oxytocin and bone.

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 17ß-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.

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

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.

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

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.

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

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.

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.

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.

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

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 TSHß. 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.

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

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.

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

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 ß-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.

Effects of radial shock waves therapy on osteoblasts activities.

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.