RESUMO
Extracellular vesicles (EVs) are emerging as mediators of a range of pathological processes, including cancer. However, their role in bone metastases has been poorly explored. We investigated EV-mediated effects of osteotropic breast cancer cells (MDA-MB-231) on bone resident cells and endothelial cells. Pretreatment of osteoblasts with conditioned medium (CM) of MDA-MB-231 (MDA) cells promoted pro-osteoclastogenic and pro-angiogenic effects by osteoblast EVs (OB-EVs), as well as an increase of RANKL-positive OB-EVs. Moreover, when treating osteoblasts with MDA-EVs, we observed a reduction of their number, metabolic activity, and alkaline phosphatase (Alp) activity. MDA-EVs also reduced transcription of Cyclin D1 and of the osteoblast-differentiating genes, while enhancing the expression of the pro-osteoclastogenic factors Rankl, Lcn2, Il1b, and Il6. Interestingly, a cytokine array on CM from osteoblasts treated with MDA-EVs showed an increase of the cytokines CCL3, CXCL2, Reg3G, and VEGF, while OPG and WISP1 were downregulated. MDA-EVs contained mRNAs of genes involved in bone metabolism, as well as cytokines, including PDGF-BB, CCL3, CCL27, VEGF, and Angiopoietin 2. In line with this profile, MDA-EVs increased osteoclastogenesis and in vivo angiogenesis. Finally, intraperitoneal injection of MDA-EVs in mice revealed their ability to reach the bone microenvironment and be integrated by osteoblasts and osteoclasts. In conclusion, we showed a role for osteoblast-derived EVs and tumor cell-derived EVs in the deregulation of bone and endothelial cell physiology, thus fueling the vicious cycle induced by bone tumors. © 2019 American Society for Bone and Mineral Research.
Assuntos
Vesículas Extracelulares , Animais , Neoplasias da Mama , Linhagem Celular Tumoral , Células Endoteliais , Humanos , Camundongos , Osteoblastos , Osteoclastos , Osteócitos , Microambiente TumoralRESUMO
During mechanical unloading, endothelial cells reduce osteogenesis and increase bone resorption. Here we describe the feedback response of endothelial cells to unloaded osteoblasts. Primary endothelial cells, ex vivo mouse aortic rings and chicken egg yolk membranes were incubated with conditioned medium from mouse primary osteoblasts (OB-CM) subjected to unit gravity or simulated microgravity, to assess its effect on angiogenesis. In vivo injection of botulin toxin A (Botox) in the quadriceps and calf muscles of C57BL/6J mice was performed to mimic disuse osteoporosis. Unloaded osteoblasts showed strong upregulation of the pro-angiogenic factor, VEGF, and their conditioned medium increased in vitro endothelial cell viability, Cyclin D1 expression, migration and tube formation, ex vivo endothelial cell sprouting from aortic rings, and in ovo angiogenesis. Treatment with the VEGF blocker, avastin, prevented unloaded OB-CM-mediated in vitro and ex vivo enhancement of angiogenesis. Bone mechanical unloading by Botox treatment, known to reduce bone mass, prompted the overexpression of VEGF in osteoblasts. The cross talk between osteoblasts and endothelial cells plays a pathophysiologic role in the response of the endothelium to unloading during disuse osteoporosis. In this context, VEGF represents a prominent osteoblast factor stimulating angiogenesis.
Assuntos
Elevação dos Membros Posteriores/fisiologia , Neovascularização Fisiológica , Osteoblastos/fisiologia , Estresse Mecânico , Animais , Toxinas Botulínicas Tipo A/farmacologia , Células Cultivadas , Embrião de Galinha , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The use of selective serotonin-reuptake inhibitors (SSRIs) has been associated with an increased risk of bone fracture, raising concerns about their increasingly broader usage. This deleterious effect is poorly understood, and thus strategies to avoid this side effect remain elusive. We show here that fluoxetine (Flx), one of the most-prescribed SSRIs, acts on bone remodeling through two distinct mechanisms. Peripherally, Flx has anti-resorptive properties, directly impairing osteoclast differentiation and function through a serotonin-reuptake-independent mechanism that is dependent on intracellular Ca2+ levels and the transcription factor Nfatc1. With time, however, Flx also triggers a brain-serotonin-dependent rise in sympathetic output that increases bone resorption sufficiently to counteract its local anti-resorptive effect, thus leading to a net effect of impaired bone formation and bone loss. Accordingly, neutralizing this second mode of action through co-treatment with the ß-blocker propranolol, while leaving the peripheral effect intact, prevents Flx-induced bone loss in mice. Hence, this study identifies a dual mode of action of SSRIs on bone remodeling and suggests a therapeutic strategy to block the deleterious effect on bone homeostasis from their chronic use.
Assuntos
Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Fluoxetina/farmacologia , Osteogênese/efeitos dos fármacos , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Sistema Nervoso Simpático/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Animais , Reabsorção Óssea , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/metabolismo , Cálcio/metabolismo , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , Imagem Óptica , Propranolol/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Triptofano Hidroxilase/genética , Microtomografia por Raio-XRESUMO
Endothelial cells are spatially close to osteoblasts and regulate osteogenesis. Moreover, they are sensitive to mechanical stimuli, therefore we hypothesized that they are implicated in the regulation of bone metabolism during unloading. Conditioned media from endothelial cells (EC-CM) subjected to simulated microgravity (0.08g and 0.008g) increased osteoblast proliferation and decreased their differentiation compared to unit gravity (1g) EC-CM. Microgravity-EC-CM increased the expression of osteoblast Rankl and subsequent osteoclastogenesis, and induced the osteoblast de-differentiating factor, Lipocalin 2 (Lcn2), whose downregulation recovered osteoblast activity, decreased Rankl expression and reduced osteoclastogenesis. Microgravity-EC-CM enhanced osteoblast NO-Synthase2 (NOS2) and CycloOXygenase2 (COX2) expression. Inhibition of NOS2 or NO signaling reduced osteoblast proliferation and rescued their differentiation. Nuclear translocation of the Lcn2/NOS2 transcription factor, NF-κB, occurred in microgravity-EC-CM-treated osteoblasts and in microgravity-treated endothelial cells, alongside high expression of the NF-κB activator, IL-1ß. IL-1ß depletion and NF-κB inhibition reduced osteoblast proliferation and rescued differentiation. Lcn2 and NOS2 were incremented in ex vivo calvarias cultured in microgravity-EC-CM, and in vivo tibias and calvarias injected with microgravity-EC-CM. Furthermore, tibias of botulin A toxin-treated and tail-suspended mice, which featured unloading and decreased bone mass, showed higher expression of IL-1ß, Lcn2 and Nos2, suggesting their pathophysiologic involvement in endothelial cell-osteoblast crosstalk.
Assuntos
Interleucina-1beta/genética , Lipocalina-2/genética , Óxido Nítrico Sintase Tipo II/genética , Osteogênese/genética , Animais , Apoptose/genética , Toxinas Botulínicas Tipo A/farmacologia , Diferenciação Celular/genética , Proliferação de Células/genética , Meios de Cultivo Condicionados/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Osteoblastos/metabolismo , Tíbia/efeitos dos fármacos , Tíbia/crescimento & desenvolvimento , Ausência de PesoRESUMO
Deficiency of Receptor Activator of NF-κB Ligand (RANKL) prevents osteoclast formation causing osteopetrosis. RANKL is a membrane-bound protein cleaved into active soluble (s)RANKL by metalloproteinase 14 (MMP14). We created a bio-device that harbors primary osteoblasts, cultured on 3D hydroxyapatite scaffolds carrying immobilized MMP14 catalytic domain. Scaffolds were sealed in diffusion chambers and implanted in RANKL-deficient mice. Mice received 1 or 2 diffusion chambers, once or twice and were sacrificed after 1 or 2 months from implants. A progressive increase of body weight was observed in the implanted groups. Histological sections of tibias of non-implanted mice were negative for the osteoclast marker Tartrate-Resistant Acid Phosphatase (TRAcP), consistent with the lack of osteoclasts. In contrast, tibias excised from implanted mice showed TRAcP-positive cells in the bone marrow and on the bone surface, these latter morphologically similar to mature osteoclasts. In mice implanted with 4 diffusion chambers total, we noted the highest number and size of TRAcP-positive cells, with quantifiable eroded bone surface and significant reduction of trabecular bone volume. These data demonstrate that our bio-device delivers effective sRANKL, inducing osteoclastogenesis in RANKL-deficient mice, supporting the feasibility of an innovative experimental strategy to treat systemic cytokine deficiencies.
Assuntos
Biotecnologia/métodos , Membrana Celular/metabolismo , Sistemas de Liberação de Medicamentos , Ligante RANK/sangue , Ligante RANK/química , Animais , Reabsorção Óssea/patologia , Agregação Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Durapatita/farmacologia , Humanos , Implantes Experimentais , Pulmão/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Metaloproteinase 14 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Estrutura Terciária de Proteína , SolubilidadeRESUMO
The transcription factor FoxO1 regulates multiple physiological processes. Here, we show that FoxO1 is highly expressed in neurons of the locus coeruleus and of various sympathetic ganglions, but not in the adrenal medulla. Consistent with this pattern of expression, mice lacking FoxO1 only in sympathetic neurons (FoxO1 Dbh-/-) display a low sympathetic tone without modification of the catecholamine content in the adrenal medulla. As a result, FoxO1 Dbh-/- mice demonstrate an increased insulin secretion, improved glucose tolerance, low energy expenditure, and high bone mass. FoxO1 favors catecholamine synthesis because it is a potent regulator of the expression of Dbh that encodes the initial and rate-limiting enzyme in the synthesis of these neurotransmitters. By identifying FoxO1 as a transcriptional regulator of the sympathetic tone, these results advance our understanding of the control of some aspects of metabolism and of bone mass accrual.
RESUMO
The portrait of osteoblasts and osteocytes has been subjected to a revision, since a large body of evidence is attributing these cells amazing roles both inside and outside the bone. The osteoblast, long confined to its bone building function, is actually a very eclectic cell, actively regulating osteoclast formation and function as well as hematopoietic stem cells homeostasis. It is also an endocrine cell, affecting energy metabolism, male fertility and cognition through the release of osteocalcin, a perfect definition-fitting hormone in its uncarboxylated state. As for the osteocytes, many evidence shows that they do not merely represent the final destination of the osteoblasts, but they are instead very active cells that, besides a mechanosensorial function, actively contribute to the bone remodelling by regulating bone formation and resorption. The regulation is exerted by the production of sclerostin (SOST), which in turn inhibits osteoblast differentiation by blocking Wnt/beta-catenin pathway. At the same time, osteocytes influence bone resorption both indirectly, by producing RANKL, which stimulates osteoclastogenesis, and directly by means of a local osteolysis, which is observed especially under pathological conditions. The great versatility of both these cells reflects the complexity of the bone tissue, which has not only a structural role, but influences and is influenced by different organs, taking part in homeostatic and adaptive responses affecting the whole organism.