Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 88
Filtrar
Mais filtros

Base de dados
País/Região como assunto
Tipo de documento
Intervalo de ano de publicação
1.
Genes Dev ; 29(14): 1463-86, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26220993

RESUMO

Fibroblast growth factor (FGF) signaling pathways are essential regulators of vertebrate skeletal development. FGF signaling regulates development of the limb bud and formation of the mesenchymal condensation and has key roles in regulating chondrogenesis, osteogenesis, and bone and mineral homeostasis. This review updates our review on FGFs in skeletal development published in Genes & Development in 2002, examines progress made on understanding the functions of the FGF signaling pathway during critical stages of skeletogenesis, and explores the mechanisms by which mutations in FGF signaling molecules cause skeletal malformations in humans. Links between FGF signaling pathways and other interacting pathways that are critical for skeletal development and could be exploited to treat genetic diseases and repair bone are also explored.


Assuntos
Doenças Ósseas/genética , Osso e Ossos/embriologia , Osso e Ossos/fisiopatologia , Fatores de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais , Animais , Doenças Ósseas/terapia , Regeneração Óssea/genética , Condrogênese , Fatores de Crescimento de Fibroblastos/genética , Humanos , Minerais/metabolismo , Mutação , Osteogênese
2.
Hum Mol Genet ; 25(7): 1281-93, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26769674

RESUMO

Patients with cystic fibrosis (CF) display low bone mass and alterations in bone formation. Mice carrying the F508del genetic mutation in the cystic fibrosis conductance regulator (Cftr) gene display reduced bone formation and decreased bone mass. However, the underlying molecular mechanisms leading to these skeletal defects are unknown, which precludes the development of an efficient anti-osteoporotic therapeutic strategy. Here we report a key role for the intermediate filament protein keratin 8 (Krt8), in the osteoblast dysfunctions in F508del-Cftr mice. We found that murine and human osteoblasts express Cftr and Krt8 at low levels. Genetic studies showed that Krt8 deletion (Krt8(-/-)) in F508del-Cftr mice increased the levels of circulating markers of bone formation, corrected the expression of osteoblast phenotypic genes, promoted trabecular bone formation and improved bone mass and microarchitecture. Mechanistically, Krt8 deletion in F508del-Cftr mice corrected overactive NF-κB signaling and decreased Wnt-ß-catenin signaling induced by the F508del-Cftr mutation in osteoblasts. In vitro, treatment with compound 407, which specifically disrupts the Krt8-F508del-Cftr interaction in epithelial cells, corrected the abnormal NF-κB and Wnt-ß-catenin signaling and the altered phenotypic gene expression in F508del-Cftr osteoblasts. In vivo, short-term treatment with 407 corrected the altered Wnt-ß-catenin signaling and bone formation in F508del-Cftr mice. Collectively, the results show that genetic or pharmacologic targeting of Krt8 leads to correction of osteoblast dysfunctions, altered bone formation and osteopenia in F508del-Cftr mice, providing a therapeutic strategy targeting the Krt8-F508del-CFTR interaction to correct the abnormal bone formation and bone loss in cystic fibrosis.


Assuntos
Doenças Ósseas Metabólicas/etiologia , Fibrose Cística/complicações , Deleção de Genes , Queratina-8/genética , Osteogênese , Animais , Doenças Ósseas Metabólicas/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/fisiopatologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , NF-kappa B , Osteoblastos/metabolismo , Transdução de Sinais , Adulto Jovem , beta Catenina
3.
J Biol Chem ; 290(11): 6903-12, 2015 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-25631051

RESUMO

The α5ß1 integrin is a key fibronectin (FN) receptor that binds to RGD-containing peptides to mediate cell adhesion. We previously reported that α5ß1 integrin promotes osteogenic differentiation in mesenchymal skeletal cells (MSCs), but the underlying mechanisms are not fully understood. In this study, we determined the signaling mechanisms induced by α5ß1 integrin interaction with its high-affinity ligand CRRETAWAC in murine and human MSCs and in vivo. We show that cyclized CRRETAWAC fully displaced MSC adhesion to FN, whereas related peptides lacking the full RRET sequence produced a partial displacement, indicating that RRET acts as an RGD-like sequence that is required to antagonize FN-mediated cell adhesion. However, all peptides increased focal adhesion kinase phosphorylation, OSE2 transcriptional activity, osteoblast gene expression, and matrix mineralization in MSCs, indicating that peptide-induced α5ß1 integrin priming can promote osteogenic differentiation independently of the RRET sequence. Biochemical analyses showed that peptide-induced α5ß1 integrin priming transiently increased PI3K/Akt phosphorylation and promoted Wnt/ß-catenin transcriptional activity independently of RRET. Consistently, pharmacological inhibition of PI3K activity reduced osteoblast differentiation and abolished Wnt regulatory gene expression induced by α5ß1 integrin priming. In vivo, systemic delivery of cyclized GACRETAWACGA linked to (DSS)6 to allow delivery to bone-forming sites for 6 weeks increased serum osteocalcin levels and improved long bone mass and microarchitecture in SAMP-6 senescent osteopenic mice. The results support a mechanism whereby α5ß1 integrin priming by high-affinity ligands integrates Wnt/ß-catenin signaling to promote osteoblast differentiation independently of cell adhesion, which could be used to improve bone mass and microarchitecture in the aging skeleton.


Assuntos
Doenças Ósseas Metabólicas/tratamento farmacológico , Integrina alfa5beta1/metabolismo , Células-Tronco Mesenquimais/citologia , Oligopeptídeos/uso terapêutico , Osteoblastos/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Doenças Ósseas Metabólicas/metabolismo , Doenças Ósseas Metabólicas/patologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , beta Catenina/metabolismo
4.
J Biol Chem ; 290(29): 18009-18017, 2015 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-26060255

RESUMO

The prevalent human ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with reduced bone formation and bone loss in mice. The molecular mechanisms by which the ΔF508-CFTR mutation causes alterations in bone formation are poorly known. In this study, we analyzed the osteoblast phenotype in ΔF508-CFTR mice and characterized the signaling mechanisms underlying this phenotype. Ex vivo studies showed that the ΔF508-CFTR mutation negatively impacted the differentiation of bone marrow stromal cells into osteoblasts and the activity of osteoblasts, demonstrating that the ΔF508-CFTR mutation alters both osteoblast differentiation and function. Treatment with a CFTR corrector rescued the abnormal collagen gene expression in ΔF508-CFTR osteoblasts. Mechanistic analysis revealed that NF-κB signaling and transcriptional activity were increased in mutant osteoblasts. Functional studies showed that the activation of NF-κB transcriptional activity in mutant osteoblasts resulted in increased ß-catenin phosphorylation, reduced osteoblast ß-catenin expression, and altered expression of Wnt/ß-catenin target genes. Pharmacological inhibition of NF-κB activity or activation of canonical Wnt signaling rescued Wnt target gene expression and corrected osteoblast differentiation and function in bone marrow stromal cells and osteoblasts from ΔF508-CFTR mice. Overall, the results show that the ΔF508-CFTR mutation impairs osteoblast differentiation and function as a result of overactive NF-κB and reduced Wnt/ß-catenin signaling. Moreover, the data indicate that pharmacological inhibition of NF-κB or activation of Wnt/ß-catenin signaling can rescue the abnormal osteoblast differentiation and function induced by the prevalent ΔF508-CFTR mutation, suggesting novel therapeutic strategies to correct the osteoblast dysfunctions in cystic fibrosis.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , NF-kappa B/imunologia , Osteoblastos/citologia , Via de Sinalização Wnt , Animais , Diferenciação Celular , Células Cultivadas , Masculino , Camundongos , Osteoblastos/imunologia , Osteoblastos/patologia , beta Catenina/imunologia
5.
Cell Mol Life Sci ; 72(7): 1347-61, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25487608

RESUMO

Several metabolic, genetic and oncogenic bone diseases are characterized by defective or excessive bone formation. These abnormalities are caused by dysfunctions in the commitment, differentiation or survival of cells of the osteoblast lineage. During the recent years, significant advances have been made in our understanding of the cellular and molecular mechanisms underlying the osteoblast dysfunctions in osteoporosis, skeletal dysplasias and primary bone tumors. This led to suggest novel therapeutic approaches to correct these abnormalities such as the modulation of WNT signaling, the pharmacological modulation of proteasome-mediated protein degradation, the induction of osteoprogenitor cell differentiation, the repression of cancer cell proliferation and the manipulation of epigenetic mechanisms. This article reviews our current understanding of the major cellular and molecular mechanisms inducing osteoblastic cell abnormalities in age-related bone loss, genetic skeletal dysplasias and primary bone tumors, and discusses emerging therapeutic strategies to counteract the osteoblast abnormalities in these disorders of bone formation.


Assuntos
Doenças do Desenvolvimento Ósseo/fisiopatologia , Neoplasias Ósseas/fisiopatologia , Osteoblastos/fisiologia , Osteoporose/fisiopatologia , Transdução de Sinais , Apoptose , Doenças do Desenvolvimento Ósseo/genética , Doenças do Desenvolvimento Ósseo/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Diferenciação Celular , Humanos , Modelos Biológicos , Osteoblastos/metabolismo , Osteoporose/genética , Osteoporose/metabolismo
6.
Am J Pathol ; 184(4): 1132-1141, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24529904

RESUMO

In patients with cystic fibrosis (CF), rib and thoracic vertebral fractures can have adverse effects on lung health because the resulting pain and debilitation can impair airway clearance. The F508del mutation in the CF transmembrane conductance regulator (Cftr) gene induces an osteopenic phenotype in humans and mice. N-butyldeoxynojyrimicin (miglustat), an approved drug for treating type 1 Gaucher disease, was found to normalize CFTR-dependent chloride transport in human F508del CFTR lung cells and in nasal mucosa of F508del CF mice. Herein, we investigated whether targeting F508del-CFTR may rescue the skeletal osteopenic phenotype in murine CF. We found that oral administration of low-dose miglustat (120 mg/kg once a day for 28 days) improved bone mass and microarchitecture in the lumbar spine and femur in F508del mice. The increased bone density was associated with an increased bone formation rate and reduced bone resorption. This effect was associated with increased 17ß-estradiol but not with insulin-like growth factor 1 serum levels in miglustat-treated F508del mice. Exposure of primary F508del osteoblasts to miglustat partially restored the deficient CFTR-dependent chloride transport in these bone-forming cells. This study provides evidence that reversal of CFTR-dependent chloride transport in osteoblasts normalizes bone mass and microarchitecture in murine CF. These findings may provide a potential therapeutic strategy to prevent or correct the bone disease in patients with CF.


Assuntos
1-Desoxinojirimicina/análogos & derivados , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/complicações , Inibidores Enzimáticos/farmacologia , 1-Desoxinojirimicina/farmacologia , Animais , Células Cultivadas , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos CFTR , Mutação , Osteoblastos/metabolismo
7.
J Cell Physiol ; 229(11): 1765-75, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24664975

RESUMO

Age-related bone loss is characterized by reduced osteoblastogenesis and excessive bone marrow adipogenesis. The mechanisms governing bone marrow mesenchymal stromal cell (BMSC) differentiation into adipocytes or osteoblasts during aging are unknown. We show here that overexpressing N-cadherin (Cadh2) in osteoblasts increased BMSC adipocyte differentiation and reduced osteoblast differentiation in young transgenic (Tg) mice whereas this phenotype was fully reversed with aging. The reversed phenotype with age was associated with enhanced Wnt5a and Wnt10b expression in osteoblasts and a concomitant increase in BMSC osteogenic differentiation. Consistent with this mechanism, conditioned media from young wild type osteoblasts inhibited adipogenesis and promoted osteoblast differentiation in BMSC from old Cadh2 Tg mice, and this response was abolished by Wnt5a and Wnt10b silencing. Transplantation of BMSC from old Cadh2 Tg mice into young Tg recipients increased Wnt5a and Wnt10b expression and rescued BMSC osteogenic differentiation. In senescent osteopenic mice, blocking the CADH2-Wnt interaction using an antagonist peptide increased Wnt5a and Wnt10b expression, bone formation, and bone mass. The data indicate that Cadh2/Wnt interaction in osteoblasts regulates BMSC lineage determination, bone formation, and bone mass and suggest a therapeutic target for promoting bone formation in the aging skeleton.


Assuntos
Envelhecimento/metabolismo , Células da Medula Óssea/citologia , Caderinas/metabolismo , Linhagem da Célula , Células-Tronco Mesenquimais/citologia , Proteínas Wnt/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia , Animais , Células da Medula Óssea/metabolismo , Reabsorção Óssea/patologia , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Transgênicos , Tamanho do Órgão , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteogênese , Ligação Proteica , Transdução de Sinais , Transplante de Células-Tronco , Proteína Wnt-5a
8.
Stem Cells ; 31(7): 1340-9, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23533197

RESUMO

The identification of the molecular mechanisms controlling the degradation of regulatory proteins in mesenchymal stromal cells (MSC) may provide clues to promote MSC osteogenic differentiation and bone regeneration. Ubiquitin ligase-dependent degradation of proteins is an important process governing cell fate. In this study, we investigated the role of the E3 ubiquitin ligase c-Cbl in MSC osteoblast differentiation and identified the mechanisms involved in this effect. Using distinct shRNA targeting c-Cbl, we showed that c-Cbl silencing promotes osteoblast differentiation in murine and human MSC, as demonstrated by increased alkaline phosphatase activity, expression of phenotypic osteoblast marker genes (RUNX2, ALP, type 1 collagen), and matrix mineralization in vitro. Coimmunoprecipitation analyses showed that c-Cbl interacts with the transcription factor STAT5, and that STAT5 forms a complex with RUNX2, a master transcription factor controlling osteoblastogenesis. Silencing c-Cbl decreased c-Cbl-mediated STAT5 ubiquitination, increased STAT5 protein level and phosphorylation, and enhanced STAT5 and RUNX2 transcriptional activity. The expression of insulin like growth factor-1 (IGF-1), a target gene of STAT5, was increased by c-Cbl silencing in MSC and in bone marrow stromal cells isolated from c-Cbl deficient mice, suggesting that IGF-1 contributes to osteoblast differentiation induced by c-Cbl silencing in MSC. Consistent with these findings, pharmacological inhibition of STAT5 activity, or neutralization of IGF-1 activity, abrogated the positive effect of c-Cbl knockdown on MSC osteogenic differentiation. Taken together, the data provide a novel functional mechanism by which the ubiquitin ligase c-Cbl regulates the osteoblastic differentiation program in mesenchymal cells by controlling Cbl-mediated STAT5 degradation and activity.


Assuntos
Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Fator de Transcrição STAT5/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Diferenciação Celular/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Fator de Transcrição STAT5/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética
9.
Calcif Tissue Int ; 94(1): 46-54, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23657489

RESUMO

Direct cell-to-cell interactions via cell adhesion molecules, in particular cadherins, are critical for morphogenesis, tissue architecture, and cell sorting and differentiation. Partially overlapping, yet distinct roles of N-cadherin (cadherin-2) and cadherin-11 in the skeletal system have emerged from mouse genetics and in vitro studies. Both cadherins are important for precursor commitment to the osteogenic lineage, and genetic ablation of Cdh2 and Cdh11 results in skeletal growth defects and impaired bone formation. While Cdh11 defines the osteogenic lineage, persistence of Cdh2 in osteoblasts in vivo actually inhibits their terminal differentiation and impairs bone formation. The action of cadherins involves both cell-cell adhesion and interference with intracellular signaling, and in particular the Wnt/ß-catenin pathway. Both cadherin-2 and cadherin-11 bind to ß-catenin, thus modulating its cytoplasmic pools and transcriptional activity. Recent data demonstrate that cadherin-2 also interferes with Lrp5/6 signaling by sequestering these receptors in inactive pools via axin binding. These data extend the biologic action of cadherins in bone forming cells, and provide novel mechanisms for development of therapeutic strategies aimed at enhancing bone formation.


Assuntos
Osso e Ossos/citologia , Osso e Ossos/metabolismo , Caderinas/metabolismo , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Transdução de Sinais/fisiologia , Animais , Humanos , Osteoblastos/metabolismo
10.
J Biol Chem ; 286(27): 24443-50, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21596750

RESUMO

Human bone marrow-derived mesenchymal stromal cells (hMSCs) have the capacity to differentiate into several cell types including osteoblasts and are therefore an important cell source for bone tissue regeneration. A crucial issue is to identify mechanisms that trigger hMSC osteoblast differentiation to promote osteogenic potential. Casitas B lineage lymphoma (Cbl) is an E3 ubiquitin ligase that ubiquitinates and targets several molecules for degradation. We hypothesized that attenuation of Cbl-mediated degradation of receptor tyrosine kinases (RTKs) may promote osteogenic differentiation in hMSCs. We show here that specific inhibition of Cbl interaction with RTKs using a Cbl mutant (G306E) promotes expression of osteoblast markers (Runx2, alkaline phosphatase, type 1 collagen, osteocalcin) and increases osteogenic differentiation in clonal bone marrow-derived hMSCs and primary hMSCs. Analysis of molecular mechanisms revealed that the Cbl mutant increased PDGF receptor α and FGF receptor 2 but not EGF receptor expression in hMSCs, resulting in increased ERK1/2 and PI3K signaling. Pharmacological inhibition of FGFR or PDGFR abrogated in vitro osteogenesis induced by the Cbl mutant. The data reveal that specific inhibition of Cbl interaction with RTKs promotes the osteogenic differentiation program in hMSCs in part by decreased Cbl-mediated PDGFRα and FGFR2 ubiquitination, providing a novel mechanistic approach targeting Cbl to promote the osteogenic capacity of hMSCs.


Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular , Mutação de Sentido Incorreto , Proteína Oncogênica v-cbl/metabolismo , Osteogênese , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Substituição de Aminoácidos , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Células da Medula Óssea/citologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Oncogênica v-cbl/antagonistas & inibidores , Proteína Oncogênica v-cbl/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células Estromais/citologia , Células Estromais/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética
11.
J Cell Biochem ; 113(6): 2047-56, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22274864

RESUMO

Mesenchymal stem cells (MSC) can differentiate into osteoblasts upon activation of Wnt signaling. Identifying targets of Wnt signaling in MSC may help promote MSC osteoblast differentiation for bone regeneration. In this study, using microarray analysis we found that Wnt3a upregulates neuregulin 1 (NRG-1) during Wnt3a-induced osteoblast differentiation in primary human MSC and murine C3H10T1/2 mesenchymal cells. Western blot and qPCR analyses confirmed that NRG-1 is upregulated by Wnt3a, and that this effect was counterbalanced by decreased expression of the NRG-1 receptor ErbB3. Consistently, exogenous NRG-1 had no effect on alkaline phosphatase (ALP) activity, an early marker of osteoblast differentiation. In contrast, small interfering RNA-mediated silencing of endogenous NRG-1 increased basal and Wnt3a-induced ALP activity in MSC. We showed that short hairpin (sh) ErbB3 and Wnt3a additively increased ß-catenin transcriptional activity and ALP activity in MSC. These effects were abrogated by DKK1, indicating that cross-talk between Wnt3a and ErbB3 control MSC osteoblast differentiation via Wnt/ß-catenin signaling. Furthermore, ErbB3 silencing decreased Src expression. Pharmacological inhibition of Src signaling promoted ErbB3- and Wnt-induced ALP activity, suggestive of a role of Src signaling in the modulation of osteoblast differentiation by ErbB3 and Wnt3a. The results indicate that downregulation of ErbB3 induced by Wnt3a contributes to Wnt3a-induced early osteoblast differentiation of MSCs through increased canonical Wnt/ß-catenin signaling and decreased Src signaling.


Assuntos
Células-Tronco Mesenquimais/citologia , Neuregulina-1/biossíntese , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Receptor ErbB-3/biossíntese , Proteína Wnt3A/metabolismo , Fosfatase Alcalina/biossíntese , Androstadienos/farmacologia , Animais , Apoptose , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Regulação para Baixo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Neuregulina-1/genética , Neuregulina-1/metabolismo , Osteogênese/genética , Interferência de RNA , RNA Interferente Pequeno , Transcrição Gênica , Via de Sinalização Wnt , Proteína Wnt3A/genética , Wortmanina , beta Catenina/biossíntese , beta Catenina/genética , Quinases da Família src/metabolismo
12.
J Cell Biochem ; 113(9): 3029-38, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22566152

RESUMO

Promoting osteoblastogenesis remains a major challenge in disorders characterized by defective bone formation. We recently showed that the alpha 5 integrin subunit (ITGA5) is critically involved in human mesenchymal cell osteoblast differentiation. In this study, we determined the potential of pharmacological ITGA5 activation by a synthetic cyclic peptide (GA-CRRETAWAC-GA) on murine osteoblast differentiation and function in vitro and bone formation in vivo. Peptide-mediated activation of ITGA5 in murine C3H10T1/2 mesenchymal cells resulted in the generation of the integrin-mediated cell signals FAK and ERK1/2-MAPKs. In vitro, peptide-based activation of ITGA5 protected from cell apoptosis but did not affect cell adhesion or replication, while it enhanced the expression of the osteoblast marker genes Runx2 and type I collagen and increased extracellular matrix (ECM) mineralization as also found with bone morphogenetic protein-2 (BMP2), a standard bone anabolic factor. When injected on adult mouse cranial bone for 3 weeks, the peptide-mediated activation of ITGA5 increased bone thickness by twofold, an effect also induced by BMP2. Histomorphometric analysis showed that this anabolic effect resulted from decreased cell apoptosis and increased bone forming surfaces and bone formation rate (BFR). We conclude that pharmacological activation of ITGA5 in mesenchymal cells is effective in promoting de novo bone formation as a result of increased osteoprogenitor cell differentiation into osteoblasts and increased cell protection from apoptosis. This peptide-based approach could be used therapeutically to promote the osteogenic capacity of osteoblast progenitor cells and to induce de novo bone formation in conditions where osteoblastogenesis is compromised.


Assuntos
Integrina alfa5/metabolismo , Osteogênese/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Animais , Western Blotting , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Proliferação de Células/efeitos dos fármacos , Integrina alfa5/genética , Camundongos , Osteogênese/genética , Reação em Cadeia da Polimerase em Tempo Real
13.
Int J Cancer ; 130(11): 2514-25, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21681744

RESUMO

Bone tumors strongly influence normal tissues and stimulate bone cells for the production of cytokines supporting proliferation and abnormal survival in cancer cells. We previously reported that the proteoglycan syndecan-2 controls the activity of various cytokines and growth factors and also modulates apoptosis and response to cytotoxic agents in osteosarcoma cell lines. Here, we show that syndecan-2 has a stronger tumor suppressor activity in vivo. We identify calpain-6 as a target gene downregulated by syndecan-2 in cells and in vivo. We demonstrate that calpain-6 expression in osteosarcoma cells depends on endothelin-1, a mediator of the tumor progression in bone. Syndecan-2 overexpression alters ERK1/2, PI3K/AKT and NFκB pathways that are calpain-6-promoting signals downstream of endothelin-1. Immunohistochemical analysis shows that calpain-6 is expressed in human bone tumors and metastases. A high expression of calpain-6 was specially found in recurrent osteosarcoma. Moreover, calpain-6 levels in primary tumors were inversely related to the response to chemotherapy. Consistently, calpain-6 was increased by doxorubicin and was found to be expressed at higher levels in doxorubicin-resistant U2OS osteosarcoma-derived cells as compared to responsive cells. Inhibition of calpain-6 with shRNA resulted in decreased proliferation, increased spontaneous apoptosis and increased sensitivity to doxorubicin and also methotrexate in responsive and resistant osteosarcoma cells. Taken together, our data show that syndecan-2 exerts its pro-apoptotic function through modulation of the endothelin-1/NFκB signaling and through downregulation of calpain-6, a protective factor that contributes to abnormal cell survival. Thus, this study identifies calpain-6 as a new possible therapeutic target in chemoresistant osteosarcoma.


Assuntos
Neoplasias Ósseas/patologia , Calpaína/fisiologia , Resistencia a Medicamentos Antineoplásicos , Endotelina-1/fisiologia , Osteossarcoma/patologia , Transdução de Sinais/fisiologia , Apoptose , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral , Humanos , NF-kappa B/fisiologia , Osteossarcoma/tratamento farmacológico , Sindecana-2/fisiologia
14.
Growth Factors ; 30(2): 117-23, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22292523

RESUMO

Fibroblast growth factors (FGFs) are important molecules that control bone formation. FGF act by activating FGF receptors (FGFRs) and downstream signaling pathways that control cells of the osteoblast lineage. Recent advances have been made in the identification of FGF/FGFR signaling pathways that control osteogenesis. Indeed, studies of mouse and human models provided novel insights into the signaling pathways that control bone formation. Genomic studies also highlighted the implication of molecular targets of FGF/FGFR signaling regulating osteoblastogenesis. Recent studies further revealed the important role of crosstalks between FGF/FGFR signaling and other signaling pathways in the regulation of osteogenesis. Finally, the importance of the mechanisms modulating FGFR degradation in the control of osteoblast differentiation has been recently revealed. This short review summarizes the recently described mechanisms underlying FGF/FGFR signaling that are involved in the control of osteoblastogenesis. This knowledge may have potential therapeutic implications in skeletal disorders characterized by abnormal bone formation.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Osteogênese/efeitos dos fármacos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Osteoblastos/citologia , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia
15.
Hum Mol Genet ; 19(9): 1678-89, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20124286

RESUMO

Dysregulations of osteoblast function induced by gain-of-function genetic mutations in fibroblast growth factor receptors (FGFRs) cause premature fusion of cranial sutures in syndromic craniosynostosis. The pathogenic signaling mechanisms induced by FGFR genetic mutations in human craniosynostosis remain largely unknown. In this study, we have used microarray analysis to investigate the signaling pathways that are activated by FGFR2 mutations in Apert craniosynostosis. Transcriptomic analysis revealed that EGFR and PDGFRalpha expression is abnormally increased in human Apert calvaria osteoblasts compared with wild-type cells. Quantitative RT-PCR and western blot analyses in Apert osteoblasts and immunohistochemical analysis of Apert sutures confirmed the increased EGFR and PDGFRalpha expression in vitro and in vivo. We demonstrate that pharmacological inhibition of EGFR and PDGFR reduces the pathological upregulation of phenotypic osteoblast genes and in vitro matrix mineralization in Apert osteoblasts. Investigation of the underlying molecular mechanisms revealed that activated FGFR2 enhances EGFR and PDGFRalpha mRNA expression via activation of PKCalpha-dependent AP-1 transcriptional activity. We also show that the increased EGFR protein expression in Apert osteoblasts results in part from a post-transcriptional mechanism involving increased Sprouty2-Cbl interaction, leading to Cbl sequestration and reduced EGFR ubiquitination. These data reveal novel molecular crosstalks between activated FGFR2, EGFR and PDGFRalpha that functionally contribute to the osteoblastic dysfunction in Apert craniosynostosis, which may provide a molecular basis for novel therapeutic approaches in this severe skeletal disorder.


Assuntos
Acrocefalossindactilia/fisiopatologia , Osteoblastos/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima , Acrocefalossindactilia/genética , Acrocefalossindactilia/metabolismo , Western Blotting , Análise Mutacional de DNA , Primers do DNA/genética , Receptores ErbB , Feto , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Imunoprecipitação , Análise em Microsséries , Mutação/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Curr Osteoporos Rep ; 10(3): 190-8, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22711369

RESUMO

Skeletal health is dependent on the balance between bone resorption and formation during bone remodeling. Multiple signaling pathways play essential roles in the maintenance of skeletal integrity by positively or negatively regulating bone cells. During the last years, significant advances have been made in our understanding of the essential signaling pathways that regulate bone cell commitment, differentiation and survival. New signaling anabolic pathways triggered by parathyroid hormone, local growth factors, Wnt signaling, and calcium sensing receptor have been identified. Novel signals induced by interactions between bone cells-matrix (integrins), osteoblasts/osteocytes (cadherins, connexins), and osteoblasts/osteoclast (ephrins, Wnt-RhoA, semaphorins) have been discovered. Recent studies revealed the key pathways (MAPK, PI3K/Akt) that critically control bone cells and skeletal mass. This review summarizes the most recent knowledge on the major signaling pathways that control bone cells, and their potential impact on the development of therapeutic strategies to improve human bone health.


Assuntos
Remodelação Óssea/fisiologia , Osso e Ossos/fisiologia , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Transdução de Sinais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Humanos
17.
Proc Natl Acad Sci U S A ; 106(44): 18587-91, 2009 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-19843692

RESUMO

Adult human mesenchymal stromal cells (hMSCs) have the potential to differentiate into chondrogenic, adipogenic, or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptome analysis, we found that integrin alpha5 (ITGA5) expression is up-regulated during dexamethasone-induced osteoblast differentiation of hMSCs. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers and in vitro osteogenesis of hMSCs. Down-regulation of endogenous ITGA5 using specific shRNAs blunted osteoblast marker gene expression and osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by ITGA5 was mediated by activation of focal adhesion kinase/ERK1/2-MAPKs and PI3K signaling pathways. Remarkably, activation of endogenous ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and osteogenic capacity of hMSCs. Importantly, we demonstrated that hMSCs engineered to overexpress ITGA5 exhibited a marked increase in their osteogenic potential in vivo. Taken together, these findings not only reveal that ITGA5 is required for osteoblast differentiation of adult hMSCs but also provide a targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs. This may be used for tissue regeneration in bone disorders where the recruitment or capacity of hMSCs is compromised.


Assuntos
Diferenciação Celular , Integrina alfa5/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Células Estromais/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inativação Gênica , Humanos , Osteoblastos/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Regulação para Cima
18.
J Biol Chem ; 285(33): 25251-8, 2010 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-20554534

RESUMO

The antiosteoporotic treatment strontium ranelate (SrRan) was shown to increase bone mass and strength by dissociating bone resorption and bone formation. To identify the molecular mechanisms of action of SrRan on osteoblasts, we investigated its effects on calcineurin-NFAT (nuclear factor of activated T cells) signaling, an important calcium sensitive pathway controlling bone formation. Using murine MC3T3-E1 and primary murine osteoblasts, we demonstrate that SrRan induces NFATc1 nuclear translocation, as shown by immunocytochemical and Western blot analyses. Molecular analysis showed that SrRan increased NFATc1 transactivation in osteoblasts, an effect that was fully abrogated by the calcineurin inhibitors cyclosporin A or FK506, confirming that SrRan activates NFATc1 signaling in osteoblasts. This has functional implications because calcineurin inhibitors blunted the enhanced osteoblast replication and expression of the osteoblast phenotypic markers Runx2, alkaline phosphatase, and type I collagen induced by SrRan. We further found that SrRan increased the expression of Wnt3a and Wnt5a as well as beta-catenin transcriptional activity in osteoblasts, and these effects were abolished by calcineurin inhibitors. The Wnt inhibitors sFRP1 and DKK1 abolished SrRan-induced osteoblast gene expression. Furthermore, blunting the Wnt5a receptor Ryk or RhoA that acts downstream of Ryk abrogated cell proliferation and osteoblast gene expression induced by SrRan. These results indicate that activation of NFATc1 and downstream canonical and non-canonical Wnt signaling pathways mediate SrRan-induced osteoblastic cell replication and differentiation, which provides novel insights into the mechanisms of action of this antiosteoporotic agent in osteoblastogenesis.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , Compostos Organometálicos/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Tiofenos/farmacologia , Proteínas Wnt/metabolismo , Animais , Western Blotting , Inibidores de Calcineurina , Diferenciação Celular/genética , Linhagem Celular , Núcleo Celular/metabolismo , Células Cultivadas , Ciclosporina/farmacologia , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Fatores de Transcrição NFATC/genética , Osteoblastos/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tacrolimo/farmacologia , Transcrição Gênica/genética , Proteínas Wnt/genética , Proteína Wnt-5a , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/genética , beta Catenina/metabolismo
19.
J Cell Physiol ; 224(2): 509-15, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20432451

RESUMO

The potential of mesenchymal stem cells (MSC) to differentiate into functional bone forming cells provides an important tool for bone regeneration. The identification of factors capable of promoting osteoblast differentiation in MSCs is therefore critical to enhance the osteogenic potential of MSCs. Using microarray analysis combined with biochemical and molecular approach, we found that FGF18, a member of the FGF family, is upregulated during osteoblast differentiation induced by dexamethasone in murine MSCs. We showed that overexpression of FGF18 by lentiviral (LV) infection, or treatment of MSCs with recombinant human (rh)FGF18 increased the expression of the osteoblast specific transcription factor Runx2, and enhanced osteoblast phenotypic marker gene expression and in vitro osteogenesis. Molecular silencing using lentiviral shRNA demonstrated that downregulation of FGFR1 or FGFR2 abrogated osteoblast gene expression induced by either LV-FGF18 or rhFGF18, indicating that FGF18 enhances osteoblast differentiation in MSCs via activation of FGFR1 or FGFR2 signaling. Biochemical and pharmacological analyses showed that the induction of phenotypic osteoblast markers by LV-FGF18 is mediated by activation of ERK1/2-MAPKs and PI3K signaling in MSCs. These results reveal that FGF18 is an essential autocrine positive regulator of the osteogenic differentiation program in murine MSCs and indicate that osteogenic differentiation induced by FGF18 in MSCs is triggered by FGFR1/FGFR2-mediated ERK1/2-MAPKs and PI3K signaling.


Assuntos
Comunicação Autócrina/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Dexametasona/farmacologia , Fatores de Crescimento de Fibroblastos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Animais , Diferenciação Celular/genética , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Camundongos , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
20.
BMC Cell Biol ; 11: 44, 2010 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-20573191

RESUMO

BACKGROUND: The potential of mesenchymal stromal cells (MSCs) to differentiate into functional bone forming cells provides an important tool for bone regeneration. The identification of factors that trigger osteoblast differentiation in MSCs is therefore critical to promote the osteogenic potential of human MSCs. In this study, we used microarray analysis to identify signalling molecules that promote osteogenic differentiation in human bone marrow stroma derived MSCs. RESULTS: Microarray analysis and validation experiments showed that the expression of IGF2 and IGFBP2 was increased together with integrin alpha5 (ITGA5) during dexamethasone-induced osteoblast differentiation in human MSCs. This effect was functional since we found that IGF2 and IGFBP2 enhanced the expression of osteoblast phenotypic markers and in vitro osteogenic capacity of hMSCs. Interestingly, we showed that downregulation of endogenous ITGA5 using specific shRNA decreased IGF2 and IGFBP2 expression in hMSCs. Conversely, ITGA5 overexpression upregulated IGF2 and IGFBP2 expression in hMSCs, which indicates tight crosstalks between these molecules. Consistent with this concept, activation of endogenous ITGA5 using a specific antibody that primes the integrin, or a peptide that specifically activates ITGA5 increased IGF2 and IGFBP2 expression in hMSCs. Finally, we showed that pharmacological inhibition of FAK/ERK1/2-MAPKs or PI3K signalling pathways that are enhanced by ITGA5 activation, blunted IGF2 and IGFBP2 expression in hMSCs. CONCLUSION: The results show that ITGA5 is a key mediator of IGF2 and IGFBP2 expression that promotes osteoblast differentiation in human MSCs, and reveal that crosstalks between ITGA5 and IGF2/IGFBP2 signalling are important mechanisms that trigger osteogenic differentiation in human bone marrow derived mesenchymal stromal cells.


Assuntos
Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Integrina alfa5/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células da Medula Óssea/citologia , Células Cultivadas , Clonagem Molecular , Indução Embrionária , Inibidores Enzimáticos/farmacologia , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fator de Crescimento Insulin-Like II/genética , Integrina alfa5/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Análise em Microsséries , Osteogênese/efeitos dos fármacos , Osteogênese/genética , RNA Interferente Pequeno/genética , Células Estromais/citologia , Transgenes/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA