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1.
J Cell Physiol ; 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34224151

RESUMO

Lipocalin 2 (LCN2) is an adipokine that accomplishes several functions in diverse organs. However, its importance in muscle and physical exercise is currently unknown. We observed that following acute high-intensity exercise ("Gran Sasso d'Italia" vertical run), LCN2 serum levels were increased. The Wnt pathway antagonist, DKK1, was also increased after the run, positively correlating with LCN2, and the same was found for the cytokine Interleukin 6. We, therefore, investigated the involvement of LCN2 in muscle physiology employing an Lcn2 global knockout (Lcn2-/- ) mouse model. Lcn2-/- mice presented with smaller muscle fibres but normal muscle performance (grip strength metre) and muscle weight. At variance with wild type (WT) mice, the inflammatory cytokine Interleukin 6 was undetectable in Lcn2-/- mice at all ages. Intriguingly, Lcn2-/- mice did not lose gastrocnemius and quadriceps muscle mass and muscle performance following hindlimb suspension, while at variance with WT, they lose soleus muscle mass. In vitro, LCN2 treatment reduced the myogenic differentiation of C2C12 and primary mouse myoblasts and influenced their gene expression. Treating myoblasts with LCN2 reduced myogenesis, suggesting that LCN2 may negatively affect muscle physiology when upregulated following high-intensity exercise.

2.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298894

RESUMO

(1) The high-fat diet (HFD) of western countries has dramatic effect on the health of several organs, including the digestive tract, leading to the accumulation of fats that can also trigger a chronic inflammatory process, such as that which occurs in non-alcohol steatohepatitis. The effects of a HFD on the small intestine, the organ involved in the absorption of this class of nutrients, are still poorly investigated. (2) To address this aspect, we administered a combined HFD with sucrose (HFD w/Suc, fat: 58% Kcal) regimen (18 months) to mice and investigated the morphological and molecular changes that occurred in the wall of proximal tract of the small intestine compared to the intestine of mice fed with a standard diet (SD) (fat: 18% Kcal). (3) We found an accumulation of lipid droplets in the mucosa of HFD w/Suc-fed mice that led to a disarrangement of mucosa architecture. Furthermore, we assessed the expression of several key players involved in lipid metabolism and inflammation, such as perilipin, leptin, leptin receptor, PI3K, p-mTOR, p-Akt, and TNF-α. All these molecules were increased in HFD mice compared to the SD group. We also evaluated anti-inflammatory molecules like adiponectin, adiponectin receptor, and PPAR-γ, and observed their significant reduction in the HFD w/Suc group compared to the control. Our data are in line with the knowledge that improper eating habits present a primary harmful assault on the bowel and the entire body's health. (4) These results represent a promising starting point for future studies, helping to better understand the complex and not fully elucidated spectrum of intestinal alterations induced by the overconsumption of fat.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Sacarose na Dieta/administração & dosagem , Sacarose na Dieta/efeitos adversos , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Adiponectina/metabolismo , Animais , Comportamento Alimentar/fisiologia , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Leptina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Obesidade/patologia , PPAR gama/metabolismo , Receptores para Leptina/metabolismo
3.
Bone ; 153: 116130, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34329816

RESUMO

Extracellular Vesicles (EVs) are becoming increasingly recognized as integral signaling vehicles in several types of cancers, including bone malignancies. However, the specific mechanisms by which EVs influence osteosarcoma progression have not been fully determined. We evaluated the effects of EVs derived from the human osteosarcoma cell line MNNG/HOS (MNNG/HOS-EVs) on bone resident cells. We found that MNNG/HOS-EVs are internalized by osteoblasts and osteoclasts in vitro, with potent inhibitory effects on osteoblast metabolic activity, cell density and alkaline phosphatase activity. Consistently, MNNG/HOS-EVs reduced the expression of cell cycle and pro-osteoblastogenic genes, whilst increasing transcriptional expression and protein release of pro-osteoclastogenic/inflammatory cytokines (RankL, Il1b, Il6 and Lcn2), pro-tumoral cytokines (CCL2,5,6,12 and CXCL1,2,5) and the metalloproteinase MMP3. MNNG/HOS-EVs did not induce osteoclast differentiation, while promoting in vitro and in vivo angiogenesis. Intriguingly, EVs derived from another osteosarcoma cell line (U2OS) reduced ALP activity but had no other effect on osteoblast phenotype. MNNG/HOS-EVs were also found to dramatically increase Serpin b2 expression in osteoblasts. To evaluate the significance of this finding, osteoblasts were forced to overexpress Serpin b2, which however did not affect osteoblast differentiation, while Il6 and Lcn2 mRNAs were up regulated. Overall, we shed light on the interactions of osteosarcoma EVs with the cells of the bone microenvironment, identifying key anti-osteoblastogenic, pro-inflammatory and pro-angiogenic factors that could contribute to osteosarcoma expansion.

4.
Int J Mol Sci ; 21(22)2020 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-33213099

RESUMO

Extracellular vesicles (EVs) are complex phospholipidic structures actively released by cells. EVs are recognized as powerful means of intercellular communication since they contain many signaling molecules (including lipids, proteins, and nucleic acids). In parallel, changes in epigenetic processes can lead to changes in gene function and finally lead to disease onset and progression. Recent breakthroughs have revealed the complex roles of non-coding RNAs (microRNAs (miRNAs) and long non-coding RNAs (lncRNAs)) in epigenetic regulation. Moreover, a substantial body of evidence demonstrates that non-coding RNAs can be shuttled among the cells and tissues via EVs, allowing non-coding RNAs to reach distant cells and exert systemic effects. Resident bone cells, including osteoclasts, osteoblasts, osteocytes, and endothelial cells, are tightly regulated by non-coding RNAs, and many of them can be exported from the cells to neighboring ones through EVs, triggering pathological conditions. For these reasons, researchers have also started to exploit EVs as a theranostic tool to address osteoporosis. In this review, we summarize some recent findings regarding the EVs' involvement in the fine regulation of non-coding RNAs in the context of bone metabolism and osteoporosis.


Assuntos
Osso e Ossos/metabolismo , Epigênese Genética , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Osteoporose/metabolismo , Osteoporose/terapia , RNA Longo não Codificante/metabolismo , Osso e Ossos/patologia , Vesículas Extracelulares/patologia , Humanos , Osteoporose/patologia
5.
J Bone Miner Res ; 35(2): 396-412, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31610048

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.

6.
Int J Mol Sci ; 20(19)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569680

RESUMO

Several studies have shown the importance of Extracellular Vesicles (EVs) in the intercellular communication between tumour and resident cells. Through EVs, tumour cells can trigger cell-signalling molecules and shuttle exogenous information to target cells, thus promoting spread of the disease. In fact, many processes are fuelled by EVs, such as tumour invasion and dormancy, drug-resistance, immune-surveillance escape, extravasation, extracellular matrix remodelling and metastasis. A key element is certainly the molecular profile of the shed cargo. Understanding the biochemical basis of EVs would help to predict the ability and propensity of cancer cells to metastasize a specific tissue, with the aim to target the release of EVs and to manipulate their content as a possible therapeutic approach. Moreover, EV profiling could help monitor the progression of cancer, providing a useful tool for more effective therapy. This review will focus on all the EV-mediated mentioned mechanisms in the context of both primary bone cancers and bone metastases.


Assuntos
Vesículas Extracelulares/metabolismo , Neoplasias/metabolismo , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Osso e Ossos/metabolismo , Comunicação Celular , Progressão da Doença , Humanos , Transdução de Sinais
7.
Front Immunol ; 10: 1901, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31440259

RESUMO

Extracellular vesicles are a heterogeneous group of cell-derived membranous structures, which facilitate intercellular communication. Recent studies have highlighted the importance of extracellular vesicles in bone homeostasis, as mediators of crosstalk between different bone-resident cells. Osteoblasts and osteoclasts are capable of releasing various types of extracellular vesicles that promote both osteogenesis, as well as, osteoclastogenesis, maintaining bone homeostasis. However, the contribution of immune cell-derived extracellular vesicles in bone homeostasis remains largely unknown. Recent proteomic studies showed that alarmins are abundantly present in/on macrophage-derived EVs. In this review we will describe these alarmins in the context of bone matrix regulation and discuss the potential contribution macrophage-derived EVs may have in this process.


Assuntos
Alarminas/metabolismo , Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Vesículas Extracelulares/metabolismo , Homeostase/fisiologia , Macrófagos/metabolismo , Animais , Comunicação Celular/fisiologia , Vesículas Extracelulares/fisiologia , Humanos , Macrófagos/fisiologia , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Osteoclastos/metabolismo , Osteoclastos/fisiologia , Osteogênese/fisiologia
8.
Br J Cancer ; 121(2): 157-171, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31239543

RESUMO

BACKGROUND: Recurrence after >5-year disease-free survival affects one-fifth of breast cancer patients and is the clinical manifestation of cancer cell reactivation after persistent dormancy. METHODS: We investigated cellular dormancy in vitro and in vivo using breast cancer cell lines and cell and molecular biology techniques. RESULTS: We demonstrated cellular dormancy in breast cancer bone metastasis, associated with haematopoietic stem cell (HSC) mimicry, in vivo competition for HSC engraftment and non-random distribution of dormant cells at the endosteal niche. Notch2 signal implication was demonstrated by immunophenotyping the endosteal niche-associated cancer cells and upon co-culture with sorted endosteal niche cells, which inhibited breast cancer cell proliferation in a Notch2-dependent manner. Blocking this signal by in vivo acute administration of the γ-secretase inhibitor, dibenzazepine, induced dormant cell mobilisation from the endosteal niche and colonisation of visceral organs. Sorted Notch2HIGH breast cancer cells exhibited a unique stem phenotype similar to HSCs and in vitro tumour-initiating ability in mammosphere assay. Human samples confirmed the existence of a small Notch2HIGH cell population in primary and bone metastatic breast cancers, with a survival advantage for Notch2HIGH vs Notch2LOW patients. CONCLUSIONS: Notch2 represents a key determinant of breast cancer cellular dormancy and mobilisation in the bone microenvironment.


Assuntos
Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Células-Tronco Hematopoéticas/fisiologia , Receptor Notch2/fisiologia , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Dibenzazepinas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Osteoblastos/fisiologia , Receptor Notch2/antagonistas & inibidores , Transdução de Sinais/fisiologia
9.
J Bone Miner Res ; 33(3): 517-533, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29091316

RESUMO

Extracellular vesicles (EVs) are newly appreciated regulators of tissue homeostasis and a means of intercellular communication. Reports have investigated the role of EVs and their cargoes in cellular regulation and have tried to fine-tune their biotechnological use, but to date very little is known on their function in bone biology. To investigate the relevance of EV-mediated communication between bone cells, we isolated EVs from primary mouse osteoblasts and assessed membrane integrity, size, and structure by transmission electron microscopy (TEM) and fluorescence-activated cell sorting (FACS). EVs actively shuttled loaded fluorochromes to osteoblasts, monocytes, and endothelial cells. Moreover, osteoblast EVs contained mRNAs shared with donor cells. Osteoblasts are known to regulate osteoclastogenesis, osteoclast survival, and osteoclast function by the pro-osteoclastic cytokine, receptor activator of nuclear factor κ-B ligand (Rankl). Osteoblast EVs were enriched in Rankl, which increased after PTH treatment. These EVs were biologically active, supporting osteoclast survival. EVs isolated from rankl-/- osteoblasts lost this pro-osteoclastic function, indicating its Rankl-dependence. They integrated ex vivo into murine calvariae, and EV-shuttled fluorochromes were quickly taken up by the bone upon in vivo EV systemic administration. Rankl-/- mice lack the osteoclast lineage and are negative for its specific marker tartrate-resistant acid phosphatase (TRAcP). Treatment of rankl-/- mice with wild-type osteoblast EVs induced the appearance of TRAcP-positive cells in an EV density-dependent manner. Finally, osteoblast EVs internalized and shuttled anti-osteoclast drugs (zoledronate and dasatinib), inhibiting osteoclast activity in vitro and in vivo. We conclude that osteoblast EVs are involved in intercellular communication between bone cells, contribute to the Rankl pro-osteoclastic effect, and shuttle anti-osteoclast drugs, representing a potential means of targeted therapeutic delivery. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.


Assuntos
Osso e Ossos/metabolismo , Vesículas Extracelulares/metabolismo , Osteoblastos/metabolismo , Animais , Comunicação Autócrina/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Sistemas de Liberação de Medicamentos , Vesículas Extracelulares/ultraestrutura , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Osteoblastos/efeitos dos fármacos , Osteoblastos/ultraestrutura , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Ligante RANK/metabolismo , RNA/metabolismo , Distribuição Tecidual/efeitos dos fármacos
10.
Horm Mol Biol Clin Investig ; 28(1): 5-20, 2016 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-27107839

RESUMO

Bone has always been regarded as a merely structural tissue, a "hard" scaffold protecting all of its "soft" fellows, while they did the rest of the work. In the last few decades this concept has totally changed, and new findings are starting to portray bone as a very talkative tissue that is capable not only of being regulated, but also of regulating other organs. In this review we aim to discuss the endocrine regulation that bone has over whole-body homeostasis, with emphasis on energy metabolism, male fertility, cognitive functions and phosphate (Pi) metabolism. These delicate tasks are mainly carried out by two known hormones, osteocalcin (Ocn) and fibroblast growth factor 23 (FGF23) and possibly other hormones that are yet to be found. The extreme plasticity and dynamicity of bone allows a very fine tuning over the actions these hormones exert, portraying this tissue as a full-fledged endocrine organ, in addition to its classical roles. In conclusion, our findings suggest that bone also has a "soft side", and is daily taking care of our entire organism in ways that were unknown until the last few years.


Assuntos
Osso e Ossos/fisiologia , Sistema Endócrino/fisiologia , Animais , Humanos , Masculino
11.
Biomaterials ; 46: 58-69, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25678116

RESUMO

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 , Solubilidade
12.
J Bone Miner Res ; 30(2): 357-68, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25112732

RESUMO

Mechanical loading represents a crucial factor in the regulation of skeletal homeostasis. Its reduction causes loss of bone mass, eventually leading to osteoporosis. In a previous global transcriptome analysis performed in mouse calvarial osteoblasts subjected to simulated microgravity, the most upregulated gene compared to unit gravity condition was Lcn2, encoding the adipokine Lipocalin 2 (LCN2), whose function in bone metabolism is poorly known. To investigate the mechanoresponding properties of LCN2, we evaluated LCN2 levels in sera of healthy volunteers subjected to bed rest, and found a significant time-dependent increase of this adipokine compared to time 0. We then evaluated the in vivo LCN2 regulation in mice subjected to experimentally-induced mechanical unloading by (1) tail suspension, (2) muscle paralysis by botulin toxin A (Botox), or (3) genetically-induced muscular dystrophy (MDX mice), and observed that Lcn2 expression was upregulated in the long bones of all of them, whereas physical exercise counteracted this increase. Mechanistically, in primary osteoblasts transfected with LCN2-expression-vector (OBs-Lcn2) we observed that Runx2 and its downstream genes, Osterix and Alp, were transcriptionally downregulated, and alkaline phosphatase (ALP) activity was less prominent versus empty-vector transduced osteoblasts (OBs-empty). OBs-Lcn2 also exhibited an increase of the Rankl/Opg ratio and IL-6 mRNA, suggesting that LCN2 could link poor differentiation of osteoblasts to enhanced osteoclast stimulation. In fact, incubation of purified mouse bone marrow mononuclear cells with conditioned media from OBs-Lcn2 cultures, or their coculture with OBs-Lcn2, improved osteoclastogenesis compared to OBs-empty, whereas treatment with recombinant LCN2 had no effect. In conclusion, our data indicate that LCN2 is a novel osteoblast mechanoresponding gene and that its regulation could be central to the pathological response of the bone tissue to low mechanical forces.


Assuntos
Proteínas de Fase Aguda/genética , Osso e Ossos/fisiopatologia , Regulação da Expressão Gênica , Homeostase/genética , Lipocalinas/genética , Mecanotransdução Celular/genética , Proteínas Oncogênicas/genética , Proteínas Proto-Oncogênicas/genética , Adulto , Animais , Repouso em Cama , Diferenciação Celular , Modelos Animais de Doenças , Células HEK293 , Elevação dos Membros Posteriores , Humanos , Lipocalina-2 , Lipocalinas/sangue , Masculino , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese , Paralisia/patologia , Paralisia/fisiopatologia , Fenótipo , Proteínas Proto-Oncogênicas/sangue
13.
Arch Biochem Biophys ; 561: 13-21, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25282390

RESUMO

Much has been written recently on osteoclast biology, but this cell type still astonishes scientists with its multifaceted functions and unique properties. The last three decades have seen a change in thinking about the osteoclast, from a cell with a single function, which just destroys the tissue it belongs to, to an "orchestrator" implicated in the concerted regulation of bone turnover. Osteoclasts have unique morphological features, organelle distribution and plasma membrane domain organization. They require polarization to cause extracellular bone breakdown and release of the digested bone matrix products into the circulation. Osteoclasts contribute to the control of skeletal growth and renewal. Alongside other organs, including kidney, gut, thyroid and parathyroid glands, they also affect calcemia and phosphatemia. Osteoclasts are very sensitive to pro-inflammatory stimuli, and studies in the '00s ascertained their tight link with the immune system, bringing about the question why bone needs a cell regulated by the immune system to remove the extracellular matrix components. Recently, osteoclasts have been demonstrated to contribute to the hematopoietic stem cell niche, controlling local calcium concentration and regulating the turnover of factors essential for hematopoietic stem cell mobilization. Finally, osteoclasts are important regulators of osteoblast activity and angiogenesis, both by releasing factors stored in the bone matrix, and secreting "clastokines" that regulate the activity of neighboring cells. All these facets will be discussed in this review article, with the aim of underscoring The Great Beauty of the osteoclast.


Assuntos
Reabsorção Óssea/patologia , Reabsorção Óssea/fisiopatologia , Osso e Ossos/patologia , Osso e Ossos/fisiopatologia , Calcificação Fisiológica , Osteoclastos/metabolismo , Osteoclastos/patologia , Animais , Humanos
14.
Arch Biochem Biophys ; 558: 70-8, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-24976175

RESUMO

Much has been written recently on osteoclast biology, but this cell type still astonishes scientists with its multifaceted functions and unique properties. The last three decades have seen a change in thinking about the osteoclast, from a cell with a single function, which just destroys the tissue it belongs to, to an "orchestrator" implicated in the concerted regulation of bone turnover. Osteoclasts have unique morphological features, organelle distribution and plasma membrane domain organization. They require polarization to cause extracellular bone breakdown and release of the digested bone matrix products into the circulation. Osteoclasts contribute to the control of skeletal growth and renewal. Alongside other organs, including kidney, gut, thyroid and parathyroid glands, they also affect calcemia and phosphatemia. Osteoclasts are very sensitive to pro-inflammatory stimuli, and studies in the '00s ascertained their tight link with the immune system, bringing about the question why bone needs a cell regulated by the immune system to remove the extracellular matrix components. Recently, osteoclasts have been demonstrated to contribute to the hematopoietic stem cell niche, controlling local calcium concentration and regulating the turnover of factors essential for hematopoietic stem cell mobilization. Finally, osteoclasts are important regulators of osteoblast activity and angiogenesis, both by releasing factors stored in the bone matrix, and secreting "clastokines" that regulate the activity of neighboring cells. All these facets will be discussed in this review article, with the aim of underscoring The Great Beauty of the osteoclast.


Assuntos
Osteoclastos , Animais , Reabsorção Óssea/patologia , Cálcio/metabolismo , Homeostase , Humanos , Imunidade , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Fosfatos/metabolismo
15.
Nat Commun ; 3: 630, 2012 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-22252554

RESUMO

Interleukin-6 (IL-6) and c-Src impair osteoblast maturation in vitro and in vivo. Given the similar effects of these factors, they are likely to establish a functional loop to maintain osteoblasts in a less mature status. Here we describe a pathway whereby c-Src stimulates IL-6 expression through the STAT3 factor, which, in response to IL-6 induces insulin-like growth factor 5 (IGFBP5), a c-Src activating factor that amplifies this loop only in immature osteoblasts. In contrast, in mature osteoblasts, IGFBP5 is enhanced by Runx2, but is no longer able to stimulate c-Src activation, as this tyrosine kinase at this stage is downregulated. We find that the IGFBP5 produced by osteoblasts stimulates osteoclastogenesis and bone resorption, acting as an osteoblast-osteoclast coupling factor. Finally, we demonstrate that the integrated actions of c-Src, IL-6 and IGFBP5 also have a role in vivo. We conclude that this pathway is relevant for bone metabolism, both in physiological and in pathological conditions.


Assuntos
Regulação da Expressão Gênica , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Interleucina-6/metabolismo , Osteoblastos/citologia , Proteínas Tirosina Quinases/metabolismo , Animais , Osso e Ossos/metabolismo , Proteína Tirosina Quinase CSK , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Meios de Cultivo Condicionados/farmacologia , Humanos , Camundongos , Modelos Biológicos , Osteoblastos/metabolismo , Interferência de RNA , Fator de Transcrição STAT3/metabolismo , Fatores de Tempo , Quinases da Família src
16.
J Bone Miner Res ; 25(1): 106-13, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20091929

RESUMO

Osteopetrosis is a genetic disease characterized by defective osteoclasts. Autosomal recessive osteopetrosis is fatal within the first years of life. Hematopoietic stem cell transplantation (HSCT) cures fewer than 50% of cases but often leaves severe neurologic damages and other dysfunctions. Osteoclast appearance after HSCT is a slow process, during which disease progression continues. We hypothesize that a support osteoclast precursor therapy may contribute to improve the osteopetrotic phenotype. To this end, we established a procedure to obtain the best yield of osteoclast precursors from human peripheral blood or mouse bone marrow mononuclear cells. These cells were injected in vivo in animal models, testing different cell injection protocols, as well as in association with CD117+ stem cells. Injected cells showed the ability to form multinucleated osteoclasts and to improve the phenotype of oc/oc osteopetrotic mice. In the best working protocol, animals presented with longer survival, improved weight and longitudinal growth, increased tibial length, tooth eruption, decreased bone volume, reduced bone marrow fibrosis, and improved hematopoiesis compared with sham-treated mice. These results provide first-hand information on the feasibility of a support osteoclast precursor therapy in osteopetrosis.


Assuntos
Osso e Ossos/patologia , Linhagem da Célula , Genes Recessivos/genética , Osteoclastos/citologia , Osteopetrose/patologia , Osteopetrose/terapia , Células-Tronco/citologia , Animais , Reabsorção Óssea/patologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Hospedeiro Imunocomprometido , Camundongos , Camundongos Endogâmicos BALB C , Osteopetrose/genética , Fenótipo , Transplante de Células-Tronco , Tíbia/patologia
17.
Bone ; 42(1): 19-29, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17936098

RESUMO

Human osteopetrosis is a rare genetic disorder caused by osteoclast failure, which ranges widely in severity. In the most severe forms, deficient bone resorption prevents enlargement of bone cavities, impairing development of bone marrow, leading to hematological failure. Closure of bone foramina causes cranial nerve compression with visual and hearing deterioration. Patients also present with osteosclerosis, short stature, malformations and brittle bones. This form is fatal in infancy, has an autosomal recessive inheritance and is cured with hematopoietic stem cell transplantation, with a rate of success <50% and unsatisfactory rescue of growth and visual deterioration. It relies on loss-of-function mutations of various genes, including the TCIRG1 gene, encoding for the a3 subunit of the H+ATPase and accounting for >50% of cases, the ClCN7 and the OSTM1 genes, which have closely related function and account for approximately 10% of cases, also presenting with neurodegeneration. Further genes are implicated in rare forms with various severities and association with other syndromes and, recently, the RANKL gene has been found to be mutated in a subset of patients lacking osteoclasts. Autosomal recessive osteopetrosis may also have intermediate severity, with a small number of cases due to loss-of-function mutations of the CAII or the PLEKHM1 genes. Dominant negative mutations of the ClCN7 gene cause the so-called Albers-Schönberg disease, which represents the most frequent and heterogeneous form of osteopetrosis, ranging from asymptomatic to intermediate/severe, thus suggesting additional genetic/environmental determinants affecting penetrance. Importantly, recent work has demonstrated that osteoblasts may also contribute to the pathogenesis of the disease, either because they are affected by intrinsic defects, or because their activity may be enhanced by deregulated osteoclasts abundantly present in most forms. Therapy is presently unsatisfactory and effort is necessary to unravel the gene defects yet unrecognized and identify new treatments to improve symptoms and save life.


Assuntos
Osteopetrose/genética , Animais , Predisposição Genética para Doença , Humanos , Modelos Animais , Mutação/genética , Osteopetrose/classificação , Osteopetrose/complicações , Osteopetrose/etiologia , Transdução de Sinais
18.
J Bone Miner Res ; 23(3): 380-91, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17997709

RESUMO

UNLABELLED: We studied phenotypic and cellular aspects in a patient with a heterozygous mutation of the PLEKHM1 gene and obtained some indications regarding the role of the protein in bone cell function. Plekhm1 is involved in osteoclast endosomal vesicle acidification and TRACP exocytosis, contributing to events involved in osteoclast-osteoblast cross-talk. INTRODUCTION: The gene PLEKHM1 encodes a nonsecretory adaptor protein that localizes to endosomal vesicles. A highly truncated Plekhm1 protein was previously found in a patient with intermediate autosomal recessive osteopetrosis. MATERIALS AND METHODS: We describe a new heterozygous mutation in the PLEKHM1 gene in a patient presenting with low vertebral and femoral T-scores and areas of focal sclerosis. Clinical evaluation, mutational analysis, assessment of in vitro osteoclast morphology and activity, transfection studies, and evaluation of osteoclast-osteoblast cross-talk were carried out. RESULTS: Direct DNA sequencing showed a heterozygous C to T substitution on cDNA position 2140 of the PLEKHM1 gene, predicted to lead to an R714C mutant protein. The mutation was not found in 104 control chromosomes. In vitro, patient's osteoclasts showed normal formation rate, morphology, number of nuclei, and actin rings but lower TRACP activity and higher endosomal pH than control osteoclasts. The patient had high serum PTH and TRACP, despite low TRACP activity in osteoclasts in vitro. HEK293 cells overexpressing either wildtype or Plekhm1-R714C showed no difference in localization of the variants, and co-transfection with a TRACP vector confirmed low TRACP activity in cells carrying the R714C mutation. RAW 264.7 osteoclast-like cells expressing the Plekhm1-R714C variant also showed low TRACP activity and reduced ability to acidify endosomal compartments compared with cells expressing the wildtype protein. Reduced intracellular TRACP was caused by increased protein secretion rather than reduced expression. TRACP-containing conditioned medium was able to increase osteoblast alkaline phosphatase, suggesting the focal osteosclerosis is a result of increased osteoclast-osteoblast coupling. CONCLUSIONS: We provide further evidence for a role of Plekhm-1 in osteoclasts by showing that a novel mutation in PLEKHM1 is associated with a complex bone phenotype of generalized osteopenia combined with "focal osteosclerosis." Our data suggest that the mutation affects endosomal acidification/maturation and TRACP exocytosis, with implications for osteoclast-osteoblast cross-talk.


Assuntos
Fosfatase Ácida/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Substituição de Aminoácidos , Endossomos/metabolismo , Exocitose , Isoenzimas/metabolismo , Glicoproteínas de Membrana/metabolismo , Osteoclastos/metabolismo , Osteopetrose/metabolismo , Fosfatase Ácida/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Proteínas Relacionadas à Autofagia , Comunicação Celular/genética , Linhagem Celular , Análise Mutacional de DNA , Endossomos/genética , Endossomos/patologia , Exocitose/genética , Feminino , Heterozigoto , Humanos , Isoenzimas/genética , Glicoproteínas de Membrana/genética , Osteoclastos/patologia , Osteopetrose/genética , Osteopetrose/patologia , Hormônio Paratireóideo/metabolismo , Mutação Puntual , Fosfatase Ácida Resistente a Tartarato
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