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1.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638536

RESUMO

Glucocorticoid-induced osteoporosis (GIO) is one of the most common secondary forms of osteoporosis. GIO is partially due to the apoptosis of osteoblasts and osteocytes. In addition, high doses of dexamethasone (DEX), a synthetic glucocorticoid receptor agonist, induces neurodegeneration by initiating inflammatory processes leading to neural apoptosis. Here, a neuroprotective bovine colostrum against glucocorticoid-induced neuronal damage was investigated for its anti-apoptotic activity in glucocorticoid-treated MC3T3-E1 osteoblastic cells. A model of apoptotic osteoblastic cells was developed by exposing MC3T3-E1 cells to DEX (0-700 µM). Colostrum co-treated with DEX was executed at 0.1-5.0 mg/mL. Cell viability was measured for all treatment schedules. Caspase-3 activation was assessed to determine both osteoblast apoptosis under DEX exposure and its potential prevention by colostrum co-treatment. Glutathione reduced (GSH) was measured to determine whether DEX-mediated oxidative stress-driven apoptosis is alleviated by colostrum co-treatment. Western blot was performed to determine the levels of p-ERK1/2, Bcl-XL, Bax, and Hsp70 proteins upon DEX or DEX plus colostrum exposure. Colostrum prevented the decrease in cell viability and the increase in caspase-3 activation and oxidative stress caused by DEX exposure. Cells, upon colostrum co-treated with DEX, exhibited higher levels of p-ERK1/2 and lower levels of Bcl-XL, Bax, and Hsp70. Our data support the notion that colostrum may be able to reduce DEX-induced apoptosis possibly via the activation of the ERK pathway and modulation of the Hsp70 system. We provided preliminary evidence on how bovine colostrum, as a complex and multi-component dairy product, in addition to its neuroprotective action, may affect osteoblastic cell survival undergoing apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Colostro/metabolismo , Fármacos Neuroprotetores/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoporose/prevenção & controle , Animais , Apoptose/fisiologia , Caspase 3/metabolismo , Bovinos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dexametasona/farmacologia , Feminino , Glucocorticoides , Glutationa/análise , Inflamação/induzido quimicamente , Camundongos , Fármacos Neuroprotetores/metabolismo , Osteoblastos/fisiologia , Osteoporose/induzido quimicamente , Estresse Oxidativo/efeitos dos fármacos , Gravidez
2.
Int J Mol Sci ; 22(19)2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34638799

RESUMO

Bone-forming cells or osteoblasts play an important role in bone modeling and remodeling processes. Osteoblast differentiation or osteoblastogenesis is orchestrated by multiple intracellular signaling pathways (e.g., bone morphogenetic proteins (BMP) and Wnt signaling pathways) and is modulated by the extracellular environment (e.g., parathyroid hormone (PTH), vitamin D, transforming growth factor ß (TGF-ß), and integrins). The regulation of bone homeostasis depends on the proper differentiation and function of osteoblast lineage cells from osteogenic precursors to osteocytes. Intracellular Ca2+ signaling relies on the control of numerous processes in osteoblast lineage cells, including cell growth, differentiation, migration, and gene expression. In addition, hyperpolarization via the activation of K+ channels indirectly promotes Ca2+ signaling in osteoblast lineage cells. An improved understanding of the fundamental physiological and pathophysiological processes in bone homeostasis requires detailed investigations of osteoblast lineage cells. This review summarizes the current knowledge on the functional impacts of K+ channels and Ca2+-permeable channels, which critically regulate Ca2+ signaling in osteoblast lineage cells to maintain bone homeostasis.


Assuntos
Canais de Cálcio/metabolismo , Osteoblastos/metabolismo , Osteogênese , Canais de Potássio/metabolismo , Transdução de Sinais , Animais , Cálcio/química , Cálcio/metabolismo , Cátions/metabolismo , Humanos , Osteoblastos/fisiologia , Potássio/química , Potássio/metabolismo
3.
Int J Mol Sci ; 22(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34638628

RESUMO

Disturbance in a differentiation program of skeletal stem cells leads to indecorous skeletogenesis. Growing evidence suggests that a fine-tuning of ubiquitin-mediated protein degradation is crucial for skeletal stem cells to maintain their stemness and osteogenic potential. Here, we demonstrate that the deubiquitinating enzyme (DUB) ubiquitin-specific protease 8 (USP8) stabilizes the Wnt receptor frizzled 5 (FZD5) by preventing its lysosomal degradation. This pathway is essential for Wnt/ß-catenin signaling and the differentiation of osteoprogenitors to mature osteoblasts. Accordingly, deletion of USP8 in osteoprogenitors (Usp8Osx) resulted in a near-complete blockade in skeletal mineralization, similar to that seen in mice with defective Wnt/ß-catenin signaling. Likewise, transplanting USP8-deficient osteoprogenitors under the renal capsule in wild-type secondary hosts did not to induce bone formation. Collectively, this study unveils an essential role for the DUB USP8 in Wnt/ß-catenin signaling in osteoprogenitors and osteogenesis during skeletal development.


Assuntos
Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Osteogênese/fisiologia , Ubiquitina Tiolesterase/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Diferenciação Celular/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Células-Tronco/metabolismo , Células-Tronco/fisiologia , beta Catenina/metabolismo
4.
Int J Mol Sci ; 22(19)2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34638677

RESUMO

Cleidocranial dysplasia (CCD), a dominantly inherited skeletal disease, is characterized by a variable phenotype ranging from dental alterations to severe skeletal defects. Either de novo or inherited mutations in the RUNX2 gene have been identified in most CCD patients. Transcription factor RUNX2, the osteogenic master gene, plays a central role in the commitment of mesenchymal stem cells to osteoblast lineage. With the aim to analyse the effects of RUNX2 mutations in CCD patients, we investigated RUNX2 gene expression and the osteogenic potential of two CCD patients' cells. In addition, with the aim to better understand how RUNX2 mutations interfere with osteogenic differentiation, we performed string analyses to identify proteins interacting with RUNX2 and analysed p53 expression levels. Our findings demonstrated for the first time that, in addition to the alteration of downstream gene expression, RUNX2 mutations impair p53 expression affecting osteogenic maturation. In conclusion, the present work provides new insights into the role of RUNX2 mutations in CCD patients and suggests that an in-depth analysis of the RUNX2-associated gene network may contribute to better understand the complex molecular and phenotypic alterations in mutant subjects.


Assuntos
Displasia Cleidocraniana/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Mutação/genética , Proteína Supressora de Tumor p53/genética , Sequência de Aminoácidos , Sequência de Bases , Diferenciação Celular/genética , Criança , Feminino , Redes Reguladoras de Genes/genética , Humanos , Masculino , Osteoblastos/fisiologia , Osteogênese/genética
5.
Cell Prolif ; 54(10): e13113, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34498342

RESUMO

OBJECTIVES: In recent years, long non-coding RNAs (lncRNAs) have been found to play a role in the occurrence, progression and prognosis of chronic musculoskeletal disorders. DESIGN AND METHODS: Literature exploring on PubMed was conducted using the combination of keywords 'LncRNA' and each of the following: 'osteoarthritis', 'rheumatoid arthritis', 'osteoporosis', 'osteogenesis', 'osteoclastogenesis', 'gout arthritis', 'Kashin-Beck disease', 'ankylosing spondylitis', 'cervical spondylotic myelopathy', 'intervertebral disc degeneration', 'human muscle disease' and 'muscle hypertrophy and atrophy'. For each disorder, we focused on the publications in the last five years (5/1/2016-2021/5/1, except for Kashin-Beck disease). Finally, we excluded publications that had been reported in reviews of various musculoskeletal disorders during the last three years. Here, we summarized the progress of research on the role of lncRNA in multiple pathological processes during musculoskeletal disorders. RESULTS: LncRNAs play a crucial role in regulating downstream gene expression and maintaining function and homeostasis of cells, especially in chondrocytes, synovial cells, osteoblasts, osteoclasts and skeletal muscle cells. CONCLUSIONS: Understanding the mechanisms of lncRNAs in musculoskeletal disorders may provide promising strategies for clinical practice.


Assuntos
Doenças Musculoesqueléticas/genética , RNA Longo não Codificante/genética , Animais , Condrócitos/fisiologia , Progressão da Doença , Homeostase/genética , Humanos , Doenças Musculoesqueléticas/patologia , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Prognóstico , Sinoviócitos/fisiologia
6.
Int J Mol Sci ; 22(17)2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34502380

RESUMO

Biophysical stimulation alters bone-forming cell activity, bone formation and remodeling. The effect of piezoelectric microvibration stimulation (PMVS) intervention on osteoporosis development remains uncertain. We investigated whether 60 Hz, 120 Hz, and 180 Hz PMVS (0.05 g, 20 min/stimulation, 3 stimulations/week for 4 consecutive weeks) intervention affected bone integrity in ovariectomized (OVX) mice or osteoblastic activity. PMVS (120 Hz)-treated OVX mice developed fewer osteoporosis conditions, including bone mineral density loss and trabecular microstructure deterioration together with decreased serum resorption marker CTX-1 levels, as compared to control OVX animals. The biomechanical strength of skeletal tissue was improved upon 120 Hz PMVS intervention. This intervention compromised OVX-induced sparse trabecular bone morphology, osteoblast loss, osteoclast overburden, and osteoclast-promoting cytokine RANKL immunostaining and reversed osteoclast inhibitor OPG immunoreactivity. Osteoblasts in OVX mice upon PMVS intervention showed strong Wnt3a immunoreaction and weak Wnt inhibitor Dkk1 immunostaining. In vitro, PMVS reversed OVX-induced loss in von Kossa-stained mineralized nodule formation, Runx2, and osteocalcin expression in primary bone-marrow stromal cells. PMVS also promoted mechanoreceptor Piezo1 expression together with increased microRNA-29a and Wnt3a expression, whereas Dkk1 rather than SOST expression was repressed in MC3T3-E1 osteoblasts. Taken together, PMVS intervention promoted Piezo1, miR-29a, and Wnt signaling to upregulate osteogenic activity and repressed osteoclastic bone resorption, delaying estrogen deficiency-induced loss in bone mass and microstructure. This study highlights a new biophysical remedy for osteoporosis.


Assuntos
Osteoblastos/metabolismo , Osteoporose/terapia , Terapia por Ultrassom/métodos , Animais , Fenômenos Biomecânicos , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Calcificação Fisiológica , Diferenciação Celular/efeitos dos fármacos , Estrogênios/metabolismo , Feminino , Canais Iônicos/metabolismo , Canais Iônicos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoblastos/fisiologia , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteoporose/metabolismo , Ovariectomia , Transdução de Sinais , Ondas Ultrassônicas , Proteína Wnt3A/metabolismo
7.
Cells ; 10(9)2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34571953

RESUMO

Despite significant advances in surgical techniques, treatment options for impaired bone healing are still limited. Inadequate bone regeneration is not only associated with pain, prolonged immobilization and often multiple revision surgeries, but also with high socioeconomic costs, underlining the importance of a detailed understanding of the bone healing process. In this regard, we previously showed that mice lacking the calcitonin receptor (CTR) display increased bone formation mediated through the increased osteoclastic secretion of sphingosine-1-phosphate (S1P), an osteoanabolic molecule promoting osteoblast function. Although strong evidence is now available for the crucial role of osteoclast-to-osteoblast coupling in normal bone hemostasis, the relevance of this paracrine crosstalk during bone regeneration is unknown. Therefore, our study was designed to test whether increased osteoclast-to-osteoblast coupling, as observed in CTR-deficient mice, may positively affect bone repair. In a standardized femoral osteotomy model, global CTR-deficient mice displayed no alteration in radiologic callus parameters. Likewise, static histomorphometry demonstrated moderate impairment of callus microstructure and normal osseous bridging of osteotomy ends. In conclusion, bone regeneration is not accelerated in CTR-deficient mice, and contrary to its osteoanabolic action in normal bone turnover, osteoclast-to-osteoblast coupling specifically involving the CTR-S1P axis, may only be of minor relevance during bone healing.


Assuntos
Regeneração Óssea/fisiologia , Osso e Ossos/metabolismo , Receptores da Calcitonina/metabolismo , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Osso e Ossos/fisiologia , Diferenciação Celular/fisiologia , Feminino , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Osteoclastos/metabolismo , Osteoclastos/fisiologia , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo
8.
Elife ; 102021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34468315

RESUMO

The central nervous system regulates activity of peripheral organs through interoception. In our previous study, we have demonstrated that PGE2/EP4 skeleton interception regulate bone homeostasis. Here, we show that ascending skeleton interoceptive signaling downregulates expression of hypothalamic neuropeptide Y (NPY) and induce lipolysis of adipose tissue for osteoblastic bone formation. Specifically, the ascending skeleton interoceptive signaling induces expression of small heterodimer partner-interacting leucine zipper protein (SMILE) in the hypothalamus. SMILE binds to pCREB as a transcriptional heterodimer on Npy promoters to inhibit NPY expression. Knockout of EP4 in sensory nerve increases expression of NPY causing bone catabolism and fat anabolism. Importantly, inhibition of NPY Y1 receptor (Y1R) accelerated oxidation of free fatty acids in osteoblasts and rescued bone loss in AvilCre:Ptger4fl/fl mice. Thus, downregulation of hypothalamic NPY expression lipolyzes free fatty acids for anabolic bone formation through a neuroendocrine descending interoceptive regulation.


Assuntos
Tecido Adiposo/metabolismo , Osso e Ossos/metabolismo , Hipotálamo/fisiologia , Interocepção/fisiologia , Neuropeptídeo Y/metabolismo , Esqueleto/fisiologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica , Metabolismo dos Lipídeos , Camundongos , Camundongos Knockout , Neuropeptídeo Y/genética , Osteoblastos/fisiologia , Transdução de Sinais
9.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361063

RESUMO

BACKGROUND: Induced tooth movement during orthodontic therapy requires mechano-induced bone remodeling. Besides various cytokines and growth-factors, neuronal guidance molecules gained attention for their roles in bone homeostasis and thus, potential roles during tooth movement. Several neuronal guidance molecules have been implicated in the regulation of bone remodeling. Amongst them, Semaphorin 3A is particular interesting as it concurrently induces osteoblast differentiation and disturbs osteoclast differentiation. METHODS: Mechano-regulation of Sema3A and its receptors PlexinA1 and Neuropilin (RT-qPCR, WB) was evaluated by applying compressive and tension forces to primary human periodontal fibroblasts (hPDLF) and alveolar bone osteoblasts (hOB). The association of the transcription factor Osterix (SP7) and SEMA3A was studied by RT-qPCR. Mechanisms involved in SEMA3A-mediated osteoblast differentiation were assessed by Rac1GTPase pull-downs, ß-catenin expression analyses (RT-qPCR) and nuclear translocation assays (IF). Osteogenic markers were analyzed by RT-qPCR. RESULTS: SEMA3A, PLXNA1 and NRP1 were differentially regulated by tension or compressive forces in hPDLF. Osterix (SP7) displayed the same pattern of regulation. Recombinant Sema3A induced the activation of Rac1GTPase, the nuclear translocation of ß-catenin and the expression of osteogenic marker genes. CONCLUSION: Sema3A, its receptors and Osterix are regulated by mechanical forces in hPDLF. SEMA3A upregulation was associated with Osterix (SP7) modulation. Sema3A-enhanced osteogenic marker gene expression in hOB might be dependent on a pathway involving Rac1GTPase and ß-catenin. Thus, Semaphorin 3A might contribute to bone remodeling during induced tooth movement.


Assuntos
Fibroblastos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neuropilinas/metabolismo , Osteoblastos/fisiologia , Ligamento Periodontal/fisiologia , Receptores de Superfície Celular/metabolismo , Semaforina-3A/metabolismo , Técnicas de Movimentação Dentária/métodos , Adolescente , Adulto , Remodelação Óssea , Diferenciação Celular , Células Cultivadas , Criança , Fibroblastos/citologia , Humanos , Proteínas do Tecido Nervoso/genética , Neuropilinas/genética , Osteoblastos/citologia , Osteogênese , Ligamento Periodontal/citologia , Receptores de Superfície Celular/genética , Semaforina-3A/genética , Adulto Jovem
10.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445293

RESUMO

In this research, we synthesize and characterize poly(glycerol sebacate) pre-polymer (pPGS) (1H NMR, FTiR, GPC, and TGA). Nano-hydroxyapatite (HAp) is synthesized using the wet precipitation method. Next, the materials are used to prepare a PGS-based composite with a 25 wt.% addition of HAp. Microporous composites are formed by means of thermally induced phase separation (TIPS) followed by thermal cross-linking (TCL) and salt leaching (SL). The manufactured microporous materials (PGS and PGS/HAp) are then subjected to imaging by means of SEM and µCT for the porous structure characterization. DSC, TGA, and water contact angle measurements are used for further evaluation of the materials. To assess the cytocompatibility and biological potential of PGS-based composites, preosteoblasts and differentiated hFOB 1.19 osteoblasts are employed as in vitro models. Apart from the cytocompatibility, the scaffolds supported cell adhesion and were readily populated by the hFOB1.19 preosteoblasts. HAp-facilitated scaffolds displayed osteoconductive properties, supporting the terminal differentiation of osteoblasts as indicated by the production of alkaline phosphatase, osteocalcin and osteopontin. Notably, the PGS/HAp scaffolds induced the production of significant amounts of osteoclastogenic cytokines: IL-1ß, IL-6 and TNF-α, which induced scaffold remodeling and promoted the reconstruction of bone tissue. Initial biocompatibility tests showed no signs of adverse effects of PGS-based scaffolds toward adult BALB/c mice.


Assuntos
Substitutos Ósseos/síntese química , Decanoatos/química , Durapatita/química , Glicerol/análogos & derivados , Polímeros/química , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Regeneração Óssea/efeitos dos fármacos , Substitutos Ósseos/química , Substitutos Ósseos/farmacologia , Substitutos Ósseos/uso terapêutico , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/fisiologia , Células Cultivadas , Feminino , Glicerol/química , Humanos , Invenções , Masculino , Teste de Materiais , Camundongos , Camundongos Endogâmicos BALB C , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteogênese/efeitos dos fármacos , Polímeros/síntese química , Porosidade , Engenharia Tecidual/tendências
11.
Elife ; 102021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34378533

RESUMO

Bone marrow adipocytes accumulate with age and in diverse disease states. However, their origins and adaptations in these conditions remain unclear, impairing our understanding of their context-specific endocrine functions and relationship with surrounding tissues. In this study, by analyzing bone and adipose tissues in the lipodystrophic 'fat-free' mouse, we define a novel, secondary adipogenesis pathway that relies on the recruitment of adiponectin-negative stromal progenitors. This pathway is unique to the bone marrow and is activated with age and in states of metabolic stress in the fat-free mouse model, resulting in the expansion of bone marrow adipocytes specialized for lipid storage with compromised lipid mobilization and cytokine expression within regions traditionally devoted to hematopoiesis. This finding further distinguishes bone marrow from peripheral adipocytes and contributes to our understanding of bone marrow adipocyte origins, adaptations, and relationships with surrounding tissues with age and disease.


Assuntos
Adipócitos/fisiologia , Adipogenia/fisiologia , Medula Óssea/fisiologia , Hematopoese/fisiologia , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/fisiologia , Fatores Etários , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Diferenciação Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/metabolismo , Osteoblastos/fisiologia
12.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206294

RESUMO

Osteoblasts, the cells that build up our skeleton, are remarkably versatile and important cells that need tight regulation in all the phases of their differentiation to guarantee proper skeletal development and homeostasis. Although we know many of the key pathways involved in osteoblast differentiation and signaling, it is becoming clearer and clearer that this is just the tip of the iceberg, and we are constantly discovering novel concepts in osteoblast physiology. In this review, we discuss well-established pathways of osteoblastic differentiation, i.e., the classical ones committing mesenchymal stromal cells to osteoblast, and then osteocytes as well as recently emerged players. In particular, we discuss micro (mi)RNAs, long non-coding (lnc)RNAs, circular (circ)RNAs, and extracellular vesicles, focusing on the mechanisms through which osteoblasts are regulated by these factors, and conversely, how they use extracellular vesicles to communicate with the surrounding microenvironment.


Assuntos
Diferenciação Celular , Osteoblastos/fisiologia , Transdução de Sinais , Animais , Vesículas Extracelulares , Humanos , MicroRNAs , Osteoblastos/metabolismo , RNA Longo não Codificante
13.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298886

RESUMO

Interaction between endothelial cells and osteoblasts is essential for bone development and homeostasis. This process is mediated in large part by osteoblast angiotropism, the migration of osteoblasts alongside blood vessels, which is crucial for the homing of osteoblasts to sites of bone formation during embryogenesis and in mature bones during remodeling and repair. Specialized bone endothelial cells that form "type H" capillaries have emerged as key interaction partners of osteoblasts, regulating osteoblast differentiation and maturation and ensuring their migration towards newly forming trabecular bone areas. Recent revolutions in high-resolution imaging methodologies for bone as well as single cell and RNA sequencing technologies have enabled the identification of some of the signaling pathways and molecular interactions that underpin this regulatory relationship. Similarly, the intercellular cross talk between endothelial cells and entombed osteocytes that is essential for bone formation, repair, and maintenance are beginning to be uncovered. This is a relatively new area of research that has, until recently, been hampered by a lack of appropriate analysis tools. Now that these tools are available, greater understanding of the molecular relationships between these key cell types is expected to facilitate identification of new drug targets for diseases of bone formation and remodeling.


Assuntos
Desenvolvimento Ósseo/fisiologia , Osso e Ossos/fisiologia , Homeostase/fisiologia , Osteoblastos/fisiologia , Animais , Remodelação Óssea/fisiologia , Células Endoteliais/fisiologia , Humanos , Osteogênese/fisiologia , Transdução de Sinais/fisiologia
14.
Cell Mol Life Sci ; 78(15): 5755-5773, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34196732

RESUMO

Cortical bone structure is a crucial determinant of bone strength, yet for many years studies of novel genes and cell signalling pathways regulating bone strength have focused on the control of trabecular bone mass. Here we focus on mechanisms responsible for cortical bone development, growth, and degeneration, and describe some recently described genetic-driven modifications in humans and mice that reveal how these processes may be controlled. We start with embryonic osteogenesis of preliminary bone structures preceding the cortex and describe how this structure consolidates then matures to a dense, vascularised cortex containing an increasing proportion of lamellar bone. These processes include modelling-induced, and load-dependent, asymmetric cortical expansion, which enables the cortex's transition from a highly porous woven structure to a consolidated and thickened highly mineralised lamellar bone structure, infiltrated by vascular channels. Sex-specific differences emerge during this process. With aging, the process of consolidation reverses: cortical pores enlarge, leading to greater cortical porosity, trabecularisation and loss of bone strength. Each process requires co-ordination between bone formation, bone mineralisation, vascularisation, and bone resorption, with a need for locational-, spatial- and cell-specific signalling pathways to mediate this co-ordination. We will discuss these processes, and a number of cell-signalling pathways identified in both murine and human genetic studies to regulate cortical bone mass, including signalling through gp130, STAT3, PTHR1, WNT16, NOTCH, NOTUM and sFRP4.


Assuntos
Desenvolvimento Ósseo/genética , Condrócitos/fisiologia , Osso Cortical/crescimento & desenvolvimento , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Osteócitos/fisiologia , Animais , Osso Cortical/fisiologia , Humanos , Porosidade , Transdução de Sinais/genética
15.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209812

RESUMO

Coupled signaling between bone-forming osteoblasts and bone-resorbing osteoclasts is crucial to the maintenance of bone homeostasis. We previously reported that v-crk avian sarcoma virus CT10 oncogene homolog-like (CrkL), which belongs to the Crk family of adaptors, inhibits bone morphogenetic protein 2 (BMP2)-mediated osteoblast differentiation, while enhancing receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation. In this study, we investigated whether CrkL can also regulate the coupling signals between osteoblasts and osteoclasts, facilitating bone homeostasis. Osteoblastic CrkL strongly decreased RANKL expression through its inhibition of runt-related transcription factor 2 (Runx2) transcription. Reduction in RANKL expression by CrkL in osteoblasts resulted in the inhibition of not only osteoblast-dependent osteoclast differentiation but also osteoclast-dependent osteoblast differentiation, suggesting that CrkL participates in the coupling signals between osteoblasts and osteoclasts via its regulation of RANKL expression. Therefore, CrkL bifunctionally regulates osteoclast differentiation through both a direct and indirect mechanism while it inhibits osteoblast differentiation through its blockade of both BMP2 and RANKL reverse signaling pathways. Collectively, these data suggest that CrkL is involved in bone homeostasis, where it helps to regulate the complex interactions of the osteoblasts, osteoclasts, and their coupling signals.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Remodelação Óssea/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos ICR , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Osteogênese/genética
16.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069142

RESUMO

Bone healing is a complex, well-organized process. Multiple factors regulate this process, including growth factors, hormones, cytokines, mechanical stimulation, and aging. One of the most important signaling pathways that affect bone healing is the Notch signaling pathway. It has a significant role in controlling the differentiation of bone mesenchymal stem cells and forming new bone. Interventions to enhance the healing of critical-sized bone defects are of great importance, and stem cell transplantations are eminent candidates for treating such defects. Understanding how Notch signaling impacts pluripotent stem cell differentiation can significantly enhance osteogenesis and improve the overall healing process upon transplantation. In Rancourt's lab, mouse embryonic stem cells (ESC) have been successfully differentiated to the osteogenic cell lineage. This study investigates the role of Notch signaling inhibition in the osteogenic differentiation of mouse embryonic and induced pluripotent stem cells (iPS). Our data showed that Notch inhibition greatly enhanced the differentiation of both mouse embryonic and induced pluripotent stem cells.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Osteogênese/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Osso e Ossos/metabolismo , Proteínas de Ciclo Celular/genética , Diferenciação Celular/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Dexametasona/farmacologia , Diaminas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Mesoderma/citologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteogênese/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Receptores Notch/metabolismo , Tiazóis/farmacologia , Fatores de Transcrição HES-1/genética , Vitamina D/farmacologia
17.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069294

RESUMO

Systemic cobalt (Co) and chromium (Cr) concentrations may be elevated in patients with metal joint replacement prostheses. Several studies have highlighted the detrimental effects of this exposure on bone cells in vitro, but the underlying mechanisms remain unclear. In this study, we use whole-genome microarrays to comprehensively assess gene expression in primary human osteoblasts, osteoclast precursors and mature resorbing osteoclasts following exposure to clinically relevant circulating versus local periprosthetic tissue concentrations of Co2+ and Cr3+ ions and CoCr nanoparticles. We also describe the gene expression response in osteoblasts on routinely used prosthesis surfaces in the presence of metal exposure. Our results suggest that systemic levels of metal exposure have no effect on osteoblasts, and primarily inhibit osteoclast differentiation and function via altering the focal adhesion and extracellular matrix interaction pathways. In contrast, periprosthetic levels of metal exposure inhibit both osteoblast and osteoclast activity by altering HIF-1α signaling and endocytic/cytoskeletal genes respectively, as well as increasing inflammatory signaling with mechanistic implications for adverse reactions to metal debris. Furthermore, we identify gene clusters and KEGG pathways for which the expression correlates with increasing Co2+:Cr3+ concentrations, and has the potential to serve as early markers of metal toxicity. Finally, our study provides a molecular basis for the improved clinical outcomes for hydroxyapatite-coated prostheses that elicit a pro-survival osteogenic gene signature compared to grit-blasted and plasma-sprayed titanium-coated surfaces in the presence of metal exposure.


Assuntos
Cromo/administração & dosagem , Cobalto/administração & dosagem , Próteses Articulares Metal-Metal , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Artroplastia de Substituição , Reabsorção Óssea/induzido quimicamente , Células Cultivadas , Cromo/toxicidade , Cobalto/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Próteses Articulares Metal-Metal/efeitos adversos , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/fisiologia , Osteoclastos/fisiologia
18.
Diabetes ; 70(7): 1419-1430, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34155042

RESUMO

Wnt signaling is an ancient and evolutionarily conserved pathway with fundamental roles in the development of adipose tissues. Roles of this pathway in mesenchymal stem cell fate determination and differentiation have been extensively studied. Indeed, canonical Wnt signaling is a significant endogenous inhibitor of adipogenesis and promoter of other cell fates, including osteogenesis, chondrogenesis, and myogenesis. However, emerging genetic evidence in both humans and mice suggests central roles for Wnt signaling in body fat distribution, obesity, and metabolic dysfunction. Herein, we highlight recent studies that have begun to unravel the contributions of various Wnt pathway members to critical adipocyte functions, including carbohydrate and lipid metabolism. We further explore compelling evidence of complex and coordinated interactions between adipocytes and other cell types within adipose tissues, including stromal, immune, and endothelial cells. Given the evolutionary conservation and ubiquitous cellular distribution of this pathway, uncovering the contributions of Wnt signaling to cell metabolism has exciting implications for therapeutic intervention in widespread pathologic states, including obesity, diabetes, and cancers.


Assuntos
Adipócitos/fisiologia , Lipogênese/fisiologia , Células-Tronco Mesenquimais/fisiologia , Via de Sinalização Wnt/fisiologia , Adipogenia/fisiologia , Animais , Humanos , Doenças Metabólicas/etiologia , Camundongos , Osteoblastos/fisiologia , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , beta Catenina/fisiologia
19.
J Orthop Surg Res ; 16(1): 359, 2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078411

RESUMO

BACKGROUND: WNT1 c.110 T>C and c.505G>T missense mutations have been identified in patients with osteogenesis imperfecta (OI). Whether these mutations affect osteoblast differentiation remains to be determined. This study aimed to investigate the effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast function, gene expression, and pathways involved in OI. METHODS: Empty vector (negative control), wild-type WNT1, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A (positive control) mutant plasmids were constructed and transfected into preosteoblast (MC3T3-E1) cells to investigate their effect on osteoblast differentiation. The expressions of osteoblast markers, including BMP2, RANKL, osteocalcin, and alkaline phosphatase (ALP), were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), enzyme-linked immunosorbent assay, and ALP staining assay, respectively. The mRNA and protein expression levels of WNT1 or the expression levels of the relevant proteins involved in the WNT1/ß-catenin signaling pathway were also determined using RT-qPCR, WB, and immunofluorescence (IF) assays after the different plasmids were transfected into MC3T3-E1 cells. RESULTS: Compared with those in the wild-type group, in the mutation groups, the mRNA and protein expression levels of BMP2 were suppressed, the expressions of osteocalcin and ALP were inhibited, and the mRNA and protein expression levels of RANKL were enhanced in MC3T3-E1 cells. WB and IF assays revealed that the protein expression levels of WNT1 in MC3T3-E1 cells were downregulated in the mutation groups compared with those in the wild-type WNT1 group. Furthermore, the expression levels of nonphosphorylated ß-catenin (non-p-ß-catenin) and phosphorylated GSK-3ß (p-GSK-3ß) were downregulated in the mutation groups compared with those in the wild-type group. However, no significant changes in the expression level of non-p-ß-catenin or p-GSK-3ß were observed in the mutation groups. CONCLUSIONS: WNT1 c.110 T>C and c.505G>T mutations may alter the proliferation and osteogenic phenotype of MC3T3-E1 linked to the progression of OI via the inhibition of the WNT1/ß-catenin signaling pathway. This is the first study to confirm the effect of WNT1 c.110 T>C and c.505G>T missense mutations on osteoblast differentiation and propose a new molecular mechanism for OI development.


Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Expressão Gênica/genética , Mutação de Sentido Incorreto/genética , Osteoblastos/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Células Cultivadas , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Osteocalcina/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo
20.
Anal Bioanal Chem ; 413(20): 5085-5093, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34169347

RESUMO

The protein heterogeneity at the single-cell level has been recognized to be vital for an understanding of various life processes during animal development. In addition, the knowledge of accurate quantity of relevant proteins at cellular level is essential for appropriate interpretation of diagnostic and therapeutic results. Some low-copy-number proteins are known to play a crucial role during cell proliferation, differentiation, and also in apoptosis. The fate decision is often based on the concentration of these proteins in the individual cells. This is likely to apply also for caspases, cysteine proteases traditionally associated with cell death via apoptosis but recently being discovered also as important factors in cell proliferation and differentiation. The hypothesis was tested in bone-related cells, where modulation of fate from apoptosis to proliferation/differentiation and vice versa is particularly challenging, e.g., towards anti-osteoporotic treatments and anti-cancer strategies. An ultrasensitive and highly selective method based on bioluminescence photon counting was used to quantify activated caspase-3/7 in order to demonstrate protein-level heterogeneity in individual cells within one population and to associate quantitative measurements with different cell fates (proliferation, differentiation, apoptosis). The results indicate a gradual increase of caspase-3/7 activation from the proliferative status to differentiation (more than three times) and towards apoptosis (more than six times). The findings clearly support one of the putative key mechanisms of non-apoptotic functions of pro-apoptotic caspases based on fine-tuning of their activation levels.


Assuntos
Caspase 3/química , Caspase 3/metabolismo , Caspase 7/química , Caspase 7/metabolismo , Osteoblastos/citologia , Animais , Apoptose , Caspase 3/genética , Caspase 7/genética , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Ativação Enzimática , Camundongos , Osteoblastos/fisiologia
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