RESUMO
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.
RESUMO
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.
Assuntos
Arginina Vasopressina/fisiologia , Densidade Óssea/fisiologia , Remodelação Óssea/fisiologia , Osteogênese/fisiologia , Ocitocina/fisiologia , Receptores de Vasopressinas/metabolismo , Sequência de Aminoácidos , Animais , Arginina Vasopressina/farmacologia , Western Blotting , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/genética , Doenças Ósseas Metabólicas/genética , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/genética , Deleção de Genes , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Osteogênese/genética , Ocitocina/farmacologia , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo , Receptores de Vasopressinas/genéticaRESUMO
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.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos/farmacologia , Aquaporina 2/metabolismo , Benzazepinas/farmacologia , Cálcio/metabolismo , Citosol/metabolismo , Receptores de Vasopressinas/metabolismo , Idoso de 80 Anos ou mais , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos/uso terapêutico , Aquaporina 2/urina , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Citosol/efeitos dos fármacos , Cães , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Síndrome de Secreção Inadequada de HAD/sangue , Síndrome de Secreção Inadequada de HAD/tratamento farmacológico , Rim/efeitos dos fármacos , Rim/metabolismo , Células Madin Darby de Rim Canino , Masculino , Camundongos Knockout , Pessoa de Meia-Idade , Osmose , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 1/metabolismo , Transporte Proteico/efeitos dos fármacos , Sódio/sangue , Tolvaptan , Água/metabolismoRESUMO
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.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Membrana Nuclear/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , Ocitocina/fisiologia , Receptores de Ocitocina/metabolismo , beta Carioferinas/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Arrestinas/antagonistas & inibidores , Arrestinas/genética , Arrestinas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/fisiologia , Ligantes , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteogênese/genética , Fosforilação , Mutação Puntual , Conformação Proteica , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/farmacologia , Receptores de Ocitocina/química , Receptores de Ocitocina/deficiência , Proteínas Recombinantes de Fusão/metabolismo , Serina/química , beta Carioferinas/antagonistas & inibidores , beta Carioferinas/genética , beta-Arrestina 1 , beta-Arrestina 2 , beta-Arrestinas , Proteínas rab5 de Ligação ao GTP/antagonistas & inibidores , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/prevenção & controle , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/prevenção & controle , Difosfonatos/farmacologia , Receptores ErbB/antagonistas & inibidores , Animais , Western Blotting , Difosfonatos/uso terapêutico , Reposicionamento de Medicamentos/métodos , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Dinâmica Molecular , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Sais de Tetrazólio , Tiazóis , Ensaio Tumoral de Célula-TroncoRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Difosfonatos/farmacologia , Receptores ErbB/antagonistas & inibidores , Modelos Moleculares , Anisotropia , Western Blotting , Linhagem Celular Tumoral , Cristalografia , Difosfonatos/metabolismo , Receptores ErbB/química , Receptores ErbB/metabolismo , Fluorescência , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Sais de Tetrazólio , TiazóisRESUMO
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.
Assuntos
Osteogênese , Receptores da Tireotropina/metabolismo , Tireotropina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Densidade Óssea , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Células Cultivadas , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Microscopia de Fluorescência , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoporose/genética , Osteoporose/metabolismo , Receptores da Tireotropina/genética , Tireotropina/farmacologia , Tireotropina Subunidade beta/metabolismo , Tireotropina Subunidade beta/farmacologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
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.
Assuntos
Arginina Vasopressina/metabolismo , Remodelação Óssea/fisiologia , Reabsorção Óssea/etiologia , Homeostase/fisiologia , Hiponatremia/complicações , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Knockout , Receptores de Vasopressinas/genéticaRESUMO
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.
Assuntos
Osso e Ossos/metabolismo , Interleucina-1/metabolismo , Sistema Nervoso Parassimpático/fisiologia , Acetilcolina/metabolismo , Animais , Apoptose , Densidade Óssea , Reabsorção Óssea , Encéfalo/metabolismo , Proliferação de Células , Coração/fisiologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Brometo de Piridostigmina/farmacologia , Transdução de SinaisRESUMO
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.
Assuntos
Anticorpos/metabolismo , Desenvolvimento Ósseo/fisiologia , Subunidade beta do Hormônio Folículoestimulante/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoporose Pós-Menopausa/prevenção & controle , Animais , Anticorpos/farmacologia , Desenvolvimento Ósseo/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Feminino , Subunidade beta do Hormônio Folículoestimulante/imunologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Knockout , Osteoclastos/citologia , Ovariectomia , Receptores do FSH/genéticaRESUMO
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.
Assuntos
Comunicação Autócrina/fisiologia , Osso e Ossos/fisiologia , Osteogênese/fisiologia , Ocitocina/fisiologia , Animais , Osso e Ossos/citologia , Estradiol/farmacologia , Estradiol/fisiologia , Humanos , Camundongos , Camundongos Knockout , Modelos Animais , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Ocitocina/deficiência , Ocitocina/genética , Receptores de Ocitocina/metabolismoRESUMO
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.
Assuntos
Osso e Ossos/metabolismo , Estrogênios/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Osteogênese/fisiologia , Ocitocina/metabolismo , Receptores de Ocitocina/metabolismo , Animais , Osso e Ossos/citologia , Feminino , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Lactação/fisiologia , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Ocitocina/genética , Receptores de Ocitocina/genéticaRESUMO
Osteoporosis, a leading cause of morbidity in the elderly, is characterized by progressive loss of bone mass resulting from excess osteoclastic bone resorption relative to osteoblastic bone formation. Here we identify Vav3, a Rho family guanine nucleotide exchange factor, as essential for stimulated osteoclast activation and bone density in vivo. Vav3-deficient osteoclasts show defective actin cytoskeleton organization, polarization, spreading and resorptive activity resulting from impaired signaling downstream of the M-CSF receptor and alpha(v)beta3 integrin. Vav3-deficient mice have increased bone mass and are protected from bone loss induced by systemic bone resorption stimuli such as parathyroid hormone or RANKL. Moreover, we provide genetic and biochemical evidence for the role of Syk tyrosine kinase as a crucial upstream regulator of Vav3 in osteoclasts. Thus, Vav3 is a potential new target for antiosteoporosis therapy.
Assuntos
Densidade Óssea , Proteínas de Ciclo Celular/fisiologia , Osteoclastos/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Reabsorção Óssea/fisiopatologia , Proteínas de Transporte/farmacologia , Proteínas de Ciclo Celular/biossíntese , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Integrina alfaVbeta3/fisiologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Glicoproteínas de Membrana/farmacologia , Camundongos , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-vav , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Fator Rho/fisiologia , Proteína-Tirosina Quinase ZAP-70RESUMO
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-kappaB 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 Ca(2+) 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.
Assuntos
Osso e Ossos/metabolismo , Ocitocina/metabolismo , Animais , Osso e Ossos/citologia , Osso e Ossos/efeitos dos fármacos , Cálcio/metabolismo , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Osteogênese/efeitos dos fármacos , Ocitocina/deficiência , Ocitocina/genética , Ocitocina/farmacologiaRESUMO
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.
Assuntos
Estrogênios/metabolismo , Osteoblastos/metabolismo , Ocitocina/biossíntese , Animais , Células Cultivadas , Humanos , Camundongos , Osteoblastos/efeitos dos fármacosRESUMO
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.
Assuntos
Células-Tronco Adultas/citologia , Osso e Ossos/citologia , Células Endoteliais/metabolismo , Fosfatase Ácida/metabolismo , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/transplante , Animais , Antígenos CD/metabolismo , Conservadores da Densidade Óssea/farmacologia , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Osso e Ossos/metabolismo , Diferenciação Celular , Linhagem da Célula , Cerâmica , Colecalciferol/farmacologia , Técnicas de Cocultura , Colágeno Tipo I/metabolismo , Células Endoteliais/citologia , Matriz Extracelular/metabolismo , Humanos , Sialoproteína de Ligação à Integrina/metabolismo , Isoenzimas/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Camundongos , Camundongos Nus , Monócitos/citologia , Monócitos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Ligante RANK/farmacologia , Fosfatase Ácida Resistente a Tartarato , Engenharia Tecidual , Alicerces TeciduaisRESUMO
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.
Assuntos
Reabsorção Óssea/etiologia , Osteoclastos/patologia , Osteoclastos/fisiologia , Voo Espacial , Ausência de Peso/efeitos adversos , Animais , Sequência de Bases , Reatores Biológicos , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Reabsorção Óssea/fisiopatologia , Bovinos , Diferenciação Celular , Células Cultivadas , DNA Complementar/genética , Expressão Gênica , Técnicas In Vitro , Camundongos , Células-Tronco/patologia , Células-Tronco/fisiologia , Simulação de Ausência de Peso/efeitos adversosRESUMO
We have reported that the posterior pituitary hormone, oxytocin (OT), known for its effects in inducing parturition, lactation and social bonding, is also a skeletal hormone. Here, we demonstrate that OT plays a key role in enabling maternal skeletal mobilization during pregnancy by enhancing the formation of bone resorbing osteoclasts. Osteoclast formation ex vivo is thus diminished in pregnant mothers with genetic OT-deficiency. OT(-/-) pups at day E20 also show a defect in trabecular bone. microCT measurements reveal normal bone volume, but increased trabecular numbers, suggesting that trabeculae in OT(-/-) pups are hypomineralized. We suggest that OT facilitates intergenerational transfer of calcium ions from a pregnant mother to the pups.
Assuntos
Osso e Ossos/embriologia , Calcificação Fisiológica/fisiologia , Osteoclastos/fisiologia , Ocitocina/fisiologia , Animais , Osso e Ossos/metabolismo , Calcificação Fisiológica/genética , Sinalização do Cálcio , Feminino , Camundongos , Camundongos Mutantes , Osteoclastos/metabolismo , Ocitocina/genética , GravidezRESUMO
The beta3 integrin cytoplasmic domain, and specifically S752, is critical for integrin localization and osteoclast (OC) function. Because growth factors such as macrophage colony-stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the beta integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors. To this end, we retrovirally expressed various beta3 integrins with cytoplasmic tail mutations in beta3-deficient OC precursors. We find that S752 in the beta3 cytoplasmic tail is required for growth factor-induced integrin activation, cytoskeletal reorganization, and membrane protrusion, thereby affecting OC adhesion, migration, and bone resorption. The small GTPases Rho and Rac mediate cytoskeletal reorganization, and activation of each is defective in OC precursors lacking a functional beta3 subunit. Activation of the upstream mediators c-Src and c-Cbl is also dependent on beta3. Interestingly, although the FAK-related kinase Pyk2 interacts with c-Src and c-Cbl, its activation is not disrupted in the absence of functional beta3. Instead, its activation is dependent upon intracellular calcium, and on the beta2 integrin. Thus, the beta3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.
Assuntos
Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Adesão Celular/genética , Citoesqueleto/metabolismo , Integrina beta3/metabolismo , Osteoclastos/metabolismo , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos/fisiologia , Animais , Reabsorção Óssea/genética , Osso e Ossos/citologia , Osso e Ossos/efeitos dos fármacos , Antígenos CD18/efeitos dos fármacos , Antígenos CD18/genética , Antígenos CD18/metabolismo , Proteína Tirosina Quinase CSK , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/genética , Quinase 2 de Adesão Focal , GTP Fosfo-Hidrolases/efeitos dos fármacos , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Substâncias de Crescimento/metabolismo , Substâncias de Crescimento/farmacologia , Integrina beta3/genética , Camundongos , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-cbl , Serina/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Quinases da Família srcRESUMO
We hypothesize that a rising follicle-stimulating hormone (FSH) level during the menopausal transition, even in the face of a normal estrogen level, contributes to increased bone resorption and profound bone loss that is accompanied by trabecular perforation and diminished bone strength. FSH has been shown to directly stimulate osteoclast formation and bone resorption, and our murine genetic studies indicate that the absence of FSH can, in part, protect against hypogonadal hyperresorption that causes bone loss. Furthermore, carefully conducted human studies, such as the Study of Women Across Nations (SWAN), indicate strong correlations between serum FSH levels and bone loss. Potential therapeutic implications include the development of antagonists to circulating FSH and its osteoclastic receptor.