RESUMO
Background: Recombinant human IGF-1/binding protein-3 (rhIGF-1/BP-3) is currently tested as a therapy in preterm infants but possible effects on the gut, including necrotizing enterocolitis (NEC), have not been tested. The aim of this study was to evaluate if rhIGF-1/BP-3 supplementation in the first days after birth negatively affects clinical variables like growth, physical activity, blood chemistry and hematology and gut maturation (e.g., intestinal permeability, morphology, enzyme activities, cytokine levels, enterocyte proliferation, NEC lesions), using NEC-sensitive preterm pigs as a model for preterm infants. Methods: Preterm pigs were given twice daily subcutaneous injections of rhIGF-1/BP-3 or vehicle. Blood was collected for IGF-1 measurements and gut tissue for NEC evaluation and biochemical analyses on day 5. Results: Baseline circulating IGF-1 levels were low in preterm pigs compared with near-term pigs reared by their mother (<20 vs. 70 ng/ml). Injection with rhIGF-1/BP-3 resulted in increased plasma IGF-1 levels for up to 6 h after injection (>40 ng/mL). rhIGF-1/BP-3 treatment reduced the incidence of severe NEC lesions (7/24 vs.16/24, p = 0.01) and overall NEC severity (1.8 ± 0.2 vs. 2.6 ± 0.3, p < 0.05, with most lesions occurring in colon). In the small intestine, villi length (405 ± 25 vs. 345 ± 33 µm) and activities of the brush border peptidases aminopeptidase N and dipeptidylpeptidase IV were increased in rhIGF-1/BP-3 treated pigs, relative to control pigs (+31-44%, both p < 0.05). The treatment had no effects on body weight, blood chemistry or hematology, except for an increase in blood leucocyte and neutrophil counts (p < 0.05, i.e., reduced neonatal neutropenia). Likewise, rhIGF-1/BP-3 treatment did not affect intestinal tissue cytokine levels (IL-1ß, IL-6, IL-8, TNFα,), enterocyte proliferation, goblet cell density, permeability or bacterial translocation to the bone marrow. Conclusion: Supplemental rhIGF-1/BP-3 did not negatively affect any of the measured variables of clinical status or gut maturation in preterm pigs. Longer-term safety and efficacy of exogenous rhIGF-1/BP-3 to support maturation of the gut and other critical organs in preterm newborns remain to be investigated in both pigs and infants.
RESUMO
Runx1, the hematopoietic lineage determining transcription factor, is present in perichondrium and chondrocytes. Here we addressed Runx1 functions, by examining expression in cartilage during mouse and human osteoarthritis (OA) progression and in response to mechanical loading. Spared and diseased compartments in knees of OA patients and in mice with surgical destabilization of the medial meniscus were examined for changes in expression of Runx1 mRNA (Q-PCR) and protein (immunoblot, immunohistochemistry). Runx1 levels were quantified in response to static mechanical compression of bovine articular cartilage. Runx1 function was assessed by cell proliferation (Ki67, PCNA) and cell type phenotypic markers. Runx1 is enriched in superficial zone (SZ) chondrocytes of normal bovine, mouse, and human tissues. Increasing loading conditions in bovine cartilage revealed a positive correlation with a significant elevation of Runx1. Runx1 becomes highly expressed at the periphery of mouse OA lesions and in human OA chondrocyte 'clones' where Runx1 co-localizes with Vcam1, the mesenchymal stem cell (MSC) marker and lubricin (Prg4), a cartilage chondroprotective protein. These OA induced cells represent a proliferative cell population, Runx1 depletion in MPCs decreases cell growth, supporting Runx1 contribution to cell expansion. The highest Runx1 levels in SZC of normal cartilage suggest a function that supports the unique phenotype of articular chondrocytes, reflected by upregulation under conditions of compression. We propose Runx1 co-expression with Vcam1 and lubricin in murine cell clusters and human 'clones' of OA cartilage, participate in a cooperative mechanism for a compensatory anabolic function.
Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoartrite do Joelho/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Bovinos , Proliferação de Células/fisiologia , Células Cultivadas , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Osteoartrite do Joelho/patologiaRESUMO
MicroRNAs (miRNAs) repress cellular protein levels to provide a sophisticated parameter of gene regulation that coordinates a broad spectrum of biological processes. Bone organogenesis is a complex process involving the differentiation and crosstalk of multiple cell types for formation and remodeling of the skeleton. Inhibition of mRNA translation by miRNAs has emerged as an important regulator of developmental osteogenic signaling pathways, osteoblast growth and differentiation, osteoclast-mediated bone resorption activity and bone homeostasis in the adult skeleton. miRNAs control multiple layers of gene regulation for bone development and postnatal functions, from the initial response of stem/progenitor cells to the structural and metabolic activity of the mature tissue. This Review brings into focus an emerging concept of bone-regulating miRNAs, the evidence for which has been gathered largely from in vivo mouse models and in vitro studies in human and mouse skeletal cell populations. Characterization of miRNAs that operate through tissue-specific transcription factors in osteoblast and osteoclast lineage cells, as well as intricate feedforward and reverse loops, has provided novel insights into the supervision of signaling pathways and regulatory networks controlling normal bone formation and turnover. The current knowledge of miRNAs characteristic of human pathologic disorders of the skeleton is presented with a future goal towards translational studies.
Assuntos
Homeostase/fisiologia , MicroRNAs/fisiologia , Osteogênese/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Humanos , Osteoblastos/fisiologiaRESUMO
The Runt-related transcription factor, Runx2, is essential for osteogenesis and is controlled by both distal (P1) and proximal (P2) promoters. To understand Runx2 function requires determination of the spatiotemporal activity of P1 and P2 to Runx2 protein production. We generated a mouse model in which the P1-derived transcript was replaced with a lacZ reporter allele, resulting in loss of P1-derived protein while simultaneously allowing discrimination between the activities of the two promoters. Loss of P1-driven expression causes developmental defects with cleidocranial dysplasia-like syndromes that persist in the postnatal skeleton. P1 activity is robust in preosteogenic mesenchyme and at the onset of bone formation but decreases as bone matures. Homozygous Runx2-P1(lacZ/lacZ) mice have a normal life span but exhibit severe osteopenia and compromised bone repair in adult mice because of osteoblastic defects and not increased osteoclastic resorption. Gene expression profiles of bone, immunohistochemical studies, and ex vivo differentiation using calvarial osteoblasts and marrow stromal cells identified mechanisms for the skeletal phenotype. The findings indicate that P1 promoter activity is necessary for generating a threshold level of Runx2 protein to commit sufficient osteoprogenitor numbers for normal bone formation. P1 promoter function is not compensated via the P2 promoter. However, the P2 transcript with compensatory mechanisms from bone morphogenetic protein (BMP) and Wnt signaling is adequate for mineralization of the bone tissue that does form. We conclude that selective utilization of the P1 and P2 promoters enables the precise spatiotemporal expression of Runx2 necessary for normal skeletogenesis and the maintenance of bone mass in the adult.
Assuntos
Calcificação Fisiológica/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Regulação da Expressão Gênica/fisiologia , Osteoblastos/metabolismo , Osteogênese/fisiologia , Regiões Promotoras Genéticas/fisiologia , Células-Tronco/metabolismo , Animais , Doenças do Desenvolvimento Ósseo/genética , Doenças do Desenvolvimento Ósseo/metabolismo , Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/metabolismo , Regeneração Óssea/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Camundongos , Camundongos TransgênicosRESUMO
MicroRNA attenuation of protein translation has emerged as an important regulator of mesenchymal cell differentiation into the osteoblast lineage. A compelling question is the extent to which miR biogenesis is obligatory for bone formation. Here we show conditional deletion of the Dicer enzyme in osteoprogenitors by Col1a1-Cre compromised fetal survival after E14.5. A mechanism was associated with the post-commitment stage of osteoblastogenesis, demonstrated by impaired ECM mineralization and reduced expression of mature osteoblast markers during differentiation of mesenchymal cells of ex vivo deleted Dicer(c/c). In contrast, in vivo excision of Dicer by Osteocalcin-Cre in mature osteoblasts generated a viable mouse with a perinatal phenotype of delayed bone mineralization which was resolved by 1 month. However, a second phenotype of significantly increased bone mass developed by 2 months, which continued up to 8 months in long bones and vertebrae, but not calvariae. Cortical bone width and trabecular thickness in Dicer(Deltaoc/Deltaoc) was twice that of Dicer(c/c) controls. Normal cell and tissue organization was observed. Expression of osteoblast and osteoclast markers demonstrated increased coupled activity of both cell types. We propose that Dicer generated miRs are essential for two periods of bone formation, to promote osteoblast differentiation before birth, and control bone accrual in the adult.
Assuntos
Diferenciação Celular , RNA Helicases DEAD-box/genética , Endorribonucleases/genética , Osteoblastos/metabolismo , Osteogênese/fisiologia , Células-Tronco/citologia , Animais , Senescência Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , RNA Helicases DEAD-box/metabolismo , Embrião de Mamíferos/metabolismo , Endorribonucleases/metabolismo , Genes Letais , Camundongos , Camundongos Endogâmicos , RNA Mensageiro/metabolismo , Ribonuclease III , Células-Tronco/metabolismoRESUMO
Inflammatory mediators and drugs which affect inflammation can influence the healing of injured tissues. Leukotrienes are potent inflammatory mediators, and similar to prostaglandins, are metabolites of arachidonic acid which can have positive or negative effects on bone and cartilage tissues. Here we tested the hypothesis that blocking the negative regulation of leukotrienes, would lead to enhanced endochondral bone formation during fracture repair. A closed femoral fracture was created in mice. Animals were divided into three groups for treatment with either montelukast sodium, a cysteinyl leukotriene type 1 receptor antagonist (trade name Singulair), zileuton, a 5-lipoxygenase enzyme inhibitor (trade name Zyflo), or carrier alone. The fractures were analyzed using radiographs, quantitative gene expression, histology and histomorphometry, and immunohistochemistry. Both the montelukast sodium group and the zileuton group exhibited enhanced fracture repair when compared with controls. Both treatment groups exhibited increased callous size and earlier bone formation when compared to controls as early as day 7. Gene expression analysis of treatment groups showed increased markers of chondrocyte proliferation and differentiation, and increased early bone formation markers when compared with controls. Treatment with montelukast sodium directly targeted the cysteinyl leukotriene type 1 receptor, leading to increased chondrocyte proliferation at early time points. These novel findings suggests a potential mechanism by which the cysteinyl leukotriene type 1 receptor acts as a negative regulator of chondrocyte proliferation, with important and previously unrecognized implications for both fracture repair, and in a broader context, systemic chondrocyte growth and differentiation.
Assuntos
Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Consolidação da Fratura/efeitos dos fármacos , Fraturas Ósseas/patologia , Antagonistas de Leucotrienos/farmacologia , Osteogênese/efeitos dos fármacos , Receptores de Leucotrienos/metabolismo , Acetatos/farmacologia , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Proliferação de Células/efeitos dos fármacos , Condrócitos/enzimologia , Ciclopropanos , Fraturas Ósseas/diagnóstico por imagem , Regulação da Expressão Gênica/efeitos dos fármacos , Hidroxiureia/análogos & derivados , Hidroxiureia/farmacologia , Hipertrofia , Leucotrieno B4/metabolismo , Camundongos , Modelos Biológicos , Quinolinas/farmacologia , Radiografia , SulfetosRESUMO
Genetic studies have identified a high bone mass of phenotype in both human and mouse when canonical Wnt signaling is increased. Secreted frizzled related protein 1 (sFRP1) is one of several Wnt antagonists and among the loss-of-function mouse models in which 32-week-old mice exhibit a high bone mass phenotype. Here we show that impact fracture healing is enhanced in this mouse model of increased Wnt signaling at a physiologic level in young (8 weeks) sFRP1(-/-) mice which do not yet exhibit significant increases in BMD. In vivo deletion of sFRP1 function improves fracture repair by promoting early bone union without adverse effects on the quality of bone tissue reflected by increased mechanical strength. We observe a dramatic reduction of the cartilage callous, increased intramembranous bone formation with bone bridging by 14 days, and early bone remodeling during the 28-day fracture repair process in the sFRP1(-/-) mice. Our molecular analyses of gene markers indicate that the effect of sFRP1 loss-of-function during fracture repair is to accelerate bone healing after formation of the initial hematoma by directing mesenchymal stem cells into the osteoblast lineage via the canonical pathway. Further evidence to support this conclusion is the observation of maximal sFRP1 levels in the cartilaginous callus of a WT mouse. Hence sFRP1(-/-) mouse progenitor cells are shifted directly into the osteoblast lineage. Thus, developing an antagonist to specifically inhibit sFRP1 represents a safe target for stimulating fracture repair and bone formation in metabolic bone disorders, osteoporosis and aging.
Assuntos
Remodelação Óssea , Consolidação da Fratura , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Tíbia/metabolismo , Fraturas da Tíbia/metabolismo , Animais , Remodelação Óssea/genética , Calo Ósseo/metabolismo , Calo Ósseo/fisiopatologia , Cartilagem/metabolismo , Cartilagem/fisiopatologia , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Modelos Animais de Doenças , Consolidação da Fratura/genética , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Knockout , Osteoblastos/metabolismo , Radiografia , Transdução de Sinais , Tíbia/diagnóstico por imagem , Tíbia/fisiopatologia , Fraturas da Tíbia/patologia , Fraturas da Tíbia/fisiopatologia , Fatores de Tempo , Proteínas Wnt/metabolismoRESUMO
Cleidocranial dysplasia (CCD) in humans is an autosomal-dominant skeletal disease that results from mutations in the bone-specific transcription factor RUNX2 (CBFA1/AML3). However, distinct RUNX2 mutations in CCD do not correlate with the severity of the disease. Here we generated a new mouse model with a hypomorphic Runx2 mutant allele (Runx2(neo7)), in which only part of the transcript is processed to full-length (wild-type) Runx2 mRNA. Homozygous Runx2(neo7/neo7) mice express a reduced level of wild-type Runx2 mRNA (55-70%) and protein. This mouse model allowed us to establish the minimal requirement of functional Runx2 for normal bone development. Runx2(neo7/neo7) mice have grossly normal skeletons with no abnormalities observed in the growth plate, but do exhibit developmental defects in calvaria and clavicles that persist through post-natal growth. Clavicle defects are caused by disrupted endochondral bone formation during embryogenesis. These hypomorphic mice have altered calvarial bone volume, as observed by histology and microCT imaging, and decreased expression of osteoblast marker genes. The bone phenotype of the heterozygous mice, which have 79-84% of wild-type Runx2 mRNA, is normal. These results show there is a critical gene dosage requirement of functional Runx2 for the formation of intramembranous bone tissues during embryogenesis. A decrease to 70% of wild-type Runx2 levels results in the CCD syndrome, whereas levels >79% produce a normal skeleton. Our findings suggest that the range of bone phenotypes in CCD patients is attributable to quantitative reduction in the functional activity of RUNX2.
Assuntos
Displasia Cleidocraniana/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Animais , Desenvolvimento Ósseo , Células Cultivadas , Clavícula/anormalidades , Clavícula/embriologia , Clavícula/crescimento & desenvolvimento , Clavícula/metabolismo , Displasia Cleidocraniana/embriologia , Displasia Cleidocraniana/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Feminino , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/metabolismo , Fenótipo , Crânio/anormalidades , Crânio/embriologia , Crânio/crescimento & desenvolvimento , Crânio/metabolismoRESUMO
Runx2, required for bone formation, is ectopically expressed in breast cancer cells. To address the mechanism by which Runx2 contributes to the osteolytic disease induced by MDA-MB-231 cells, we investigated the effect of Runx2 on key components of the "vicious cycle" of transforming growth factor beta (TGFbeta)-mediated tumor growth and osteolysis. We find that Runx2 directly up-regulates Indian Hedgehog (IHH) and colocalizes with Gli2, a Hedgehog signaling molecule. These events further activate parathyroid hormone-related protein (PTHrP). Furthermore, Runx2 directly regulates the TGFbeta-induced PTHrP levels. A subnuclear targeting deficient mutant Runx2, which disrupts TGFbeta-induced Runx2-Smad interactions, failed to induce IHH and downstream events. In addition, Runx2 knockdown in MDA-MB-231 inhibited IHH and PTHrP expression in the presence of TGFbeta. In vivo blockade of the Runx2-IHH pathway in MDA-MB-231 cells by Runx2 short hairpin RNA inhibition prevented the osteolytic disease. Thus, our studies define a novel role of Runx2 in up-regulating the vicious cycle of metastatic bone disease, in addition to Runx2 regulation of genes related to progression of tumor metastasis.
Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Proteínas Hedgehog/genética , Ativação Transcricional , Animais , Neoplasias Ósseas/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes bcl-1/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos SCID , Modelos Biológicos , Proteínas Nucleares/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/genética , Distribuição Tecidual , Ativação Transcricional/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Transplante Heterólogo , Células Tumorais Cultivadas , Proteína Gli2 com Dedos de ZincoRESUMO
The WW domain-containing oxidoreductase (WWOX) gene encodes a tumor suppressor. We have previously shown that targeted ablation of the Wwox gene in mouse increases the incidence of spontaneous and chemically induced tumors. To investigate WWOX function in vivo, we examined Wwox-deficient (Wwox(-/-)) mice for phenotypical abnormalities. Wwox(-/-) mice are significantly reduced in size, die at the age of 2-3 weeks, and suffer a metabolic disorder that affects the skeleton. Wwox(-/-) mice exhibit a delay in bone formation from a cell autonomous defect in differentiation beginning at the mineralization stage shown in calvarial osteoblasts ex vivo and supported by significantly decreased bone formation parameters in Wwox(-/-) mice by microcomputed tomography analyses. Wwox(-/-) mice develop metabolic bone disease, as a consequence of reduced serum calcium, hypoproteinuria, and hypoglycemia leading to increased osteoclast activity and bone resorption. Interestingly, we find WWOX physically associates with RUNX2, the principal transcriptional regulator of osteoblast differentiation, and on osteocalcin chromatin. We show WWOX functionally suppresses RUNX2 transactivation ability in osteoblasts. In breast cancer MDA-MB-242 cells that lack endogenous WWOX protein, restoration of WWOX expression inhibited Runx2 and RUNX2 target genes related to metastasis. Affymetrix mRNA profiling revealed common gene targets in multiple tissues. In Wwox(-/-) mice, genes related to nucleosome assembly and cell growth genes were down-regulated, and negative regulators of skeletal metabolism exhibited increased expression. Our results demonstrate an essential requirement for the WWOX tumor suppressor in postnatal survival, growth, and metabolism and suggest a central role for WWOX in regulation of bone tissue formation.
Assuntos
Osso e Ossos/metabolismo , Regulação da Expressão Gênica , Oxirredutases/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Animais Recém-Nascidos , Diferenciação Celular , Linhagem Celular Tumoral , Cromatina/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Oxirredutases/metabolismo , Fenótipo , Proteínas Supressoras de Tumor/metabolismo , Oxidorredutase com Domínios WWRESUMO
Secreted frizzled related protein-1 (sFRP1), an antagonist of Wnt signaling, regulates cell proliferation, differentiation and apoptosis and negatively regulates bone formation. The spatial and temporal pattern of endogenous sFRP1 expression and loss-of-function were examined in the sFRP1-LacZ knock-in mouse (sFRP1-/-) during embryonic development and post-natal growth. beta-gal activity representing sFRP1 expression is robust in brain, skeleton, kidney, eye, spleen, abdomen, heart and somites in early embryos, but sFRP1 gene inactivation in these tissues did not compromise normal embryonic and post-natal development. Kidney histology revealed increased numbers of glomeruli in KO mice, observed after 5 years of breeding. In the skeleton, we show sFRP1 expression is found in relation to the mineralizing front of bone tissue during skeletal development from E15.5 to birth. Trabecular bone volume and bone mineral density in the sFRP1-/- mouse compared to WT was slightly increased during post-natal growth. Calvarial osteoblasts from newborn sFRP1-/- mice exhibited a 20% increase in cell proliferation and differentiation at the early stages of osteoblast maturation. sFRP1 expression was observed in osteoclasts, but this did not affect osteoclast number or activity. These findings have identified functions for sFRP1 in kidney and bone that are not redundant with other sFRPs. In summary, the absence of major organ abnormalities, the enhanced bone formation and a normal life span with no detection of spontaneous tumors suggests that targeting sFRP1 can be used as a therapeutic strategy for increasing bone mass in metabolic bone disorders or promoting fracture healing by modulating Wnt signaling.
Assuntos
Osso e Ossos/embriologia , Encéfalo/embriologia , Desenvolvimento Embrionário , Rim/embriologia , Proteínas/metabolismo , Animais , Northern Blotting , Densidade Óssea , Remodelação Óssea/fisiologia , Osso e Ossos/citologia , Diferenciação Celular , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Osteogênese/fisiologiaRESUMO
The osteogenic Runt-related (Runx2) transcription factor negatively regulates proliferation and ribosomal gene expression in normal diploid osteoblasts, but is up-regulated in metastatic breast and prostate cancer cells. Thus, Runx2 may function as a tumor suppressor or an oncogene depending on the cellular context. Here we show that Runx2-deficient primary osteoblasts fail to undergo senescence as indicated by the absence of beta-gal activity and p16(INK4a) tumor suppressor expression. Primary Runx2-null osteoblasts have a growth advantage and exhibit loss of p21(WAF1/CIP1) and p19(ARF) expression. Reintroduction of WT Runx2, but not a subnuclear targeting-defective mutant, induces both p21(WAF/CIP1) and p19(ARF) mRNA and protein resulting in cell-cycle inhibition. Accumulation of spontaneous phospho-H2A.X foci, loss of telomere integrity and the Mre11/Rad50/Nbs1 DNA repair complex, and a delayed DNA repair response all indicate that Runx2 deficiency leads to genomic instability. We propose that Runx2 functions as a tumor suppressor in primary diploid osteoblasts and that subnuclear targeting contributes to Runx2-mediated tumor suppression.
Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , Transformação Celular Neoplásica/metabolismo , Senescência Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Animais , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/deficiência , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Marcação de Genes , Instabilidade Genômica/fisiologia , Camundongos , Camundongos Knockout , Osteoblastos/metabolismo , Osteoblastos/patologiaRESUMO
Pocket proteins and cyclin-dependent kinase (CDK) inhibitors negatively regulate cell proliferation and can promote differentiation. However, which members of these gene families, which cell type they interact in, and what they do to promote differentiation in that cell type during mouse development are largely unknown. To identify the cell types in which p107 and p27 interact, we generated compound mutant mice. These mice were null for p107 and had a deletion in p27 that prevented its binding to cyclin-CDK complexes. Although a fraction of these animals survived into adulthood and looked similar to single p27 mutant mice, a larger number of animals died at birth or within a few weeks thereafter. These animals displayed defects in chondrocyte maturation and endochondral bone formation. Proliferation of chondrocytes was increased, and ectopic ossification was observed. Uncommitted mouse embryo fibroblasts could be induced into the chondrocytic lineage ex vivo, but these cells failed to mature normally. These results demonstrate that p27 carries out overlapping functions with p107 in controlling cell cycle exit during chondrocyte maturation. The phenotypic similarities between p107(-/-) p27(D51/D51) and p107(-/-) p130(-/-) mice and the cells derived from them suggest that p27 and p130 act in an analogous pathway during chondrocyte maturation.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Osteogênese/genética , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína p130 Retinoblastoma-Like/metabolismo , Animais , Animais Recém-Nascidos , Peso Corporal , Osso e Ossos/anatomia & histologia , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Condrócitos/citologia , Condrogênese , Inibidor de Quinase Dependente de Ciclina p27/deficiência , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/metabolismo , Masculino , Camundongos , Tamanho do Órgão , Fenótipo , Proteína p107 Retinoblastoma-Like/deficiência , Fase S , Análise de SobrevidaRESUMO
We present an overview of the concepts of tissue-specific transcriptional control mechanisms essential for development of the bone cell phenotype. BMP2 induced transcription factors constitute a network of activities and molecular switches for bone development and osteoblast differentiation. Among these regulators are Runx2 (Cbfa1/AML3), the principal osteogenic master gene for bone formation, as well as homeodomain proteins and osterix. Runx2 has multiple regulatory activities, including activation or repression of gene expression, and integration of biological signals from developmental cues, such as BMP/TGFbeta, Wnt and Src signaling pathways. Runx2 provides a new paradigm for transcriptional control by functioning as a principal scaffolding protein in nuclear microenvironments to control gene expression in response to physiologic signals (growth factors, cytokines and hormones). The protein serves as a hub for the coordination of activities essential for the expansion and differentiation of osteogenic lineage cells through the formation of co-regulatory protein complexes organized in subnuclear domains. Mechanisms by which Runx2 supports commitment to osteogenesis and determines cell fate involve its retention on mitotic chromosomes. Disruption of a unique protein module, the subnuclear targeting signal of Runx2, has profound effects on osteoblast differentiation and metastasis of cancer cells in the bone microenvironment. Runx2 target genes include regulators of cell growth control, components of the bone extracellular matrix, angiogenesis, and signaling proteins for development of the osteoblast phenotype and bone turnover. The specificity of Runx2 regulatory activities provides a basis for novel therapeutic strategies to correct bone disorders.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes , Osteoblastos/citologia , Animais , Proteínas Morfogenéticas Ósseas/fisiologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Carcinoma/genética , Carcinoma/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Humanos , Modelos Biológicos , Metástase Neoplásica/genética , Osteogênese/genética , Transdução de Sinais , Proteínas Smad/metabolismo , Proteínas Smad/fisiologia , Proteínas Wnt/fisiologiaRESUMO
Canonical Wnt signaling (beta-catenin/TCF) has emerged as a key regulator of skeletogenesis. In this study, chondrogenesis is examined in a mouse model in which the Wnt antagonist secreted frizzled related protein 1 (sFRP1) is non-functional and results in a high bone mass phenotype and activation through the canonical pathway of the Runx2 transcription factor that is essential for bone formation. We find during the period of rapid post-natal growth, shortened height of the growth plate and increased calcification of the hypertrophic zone (HZ) in the sFRP1-/- mouse, indicating accelerated endochondral ossification. Using mouse embryo fibroblasts (MEFs) induced into the chondrogenic lineage, increased chondrogenesis and accelerating differentiation of hypertrophic chondrocytes in the sFRP1-/- MEFs was observed compared to WT cells. The induced maturation of hypertrophic chondrocytes in sFRP1(-/-) MEFs was inversely correlated to phospho-beta-catenin levels, indicating involvement of activated canonical Wnt signaling characterized by an increased expression of collagen type 2a1 and Sox 9. However, an absence of Indian hedgehog expression which occurs in WT cells was found. SFRP1-/- cells also exhibited an early induction of collagen type 10a1. Thus, these modifications in gene expression are contributing mechanism(s) for increased chondrocyte differentiation in SFRP1-/- cells. These studies have identified sFRP1 as a critical negative regulator of Wnt signaling for the normal progression of chondrocyte differentiation. Microarray gene profiling provided additional novel insights into the regulatory factors for appropriate Wnt signaling necessary for the control of chondrocyte maturation.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Diferenciação Celular/fisiologia , Condrócitos/patologia , Condrócitos/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas de Membrana/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Proteínas Wnt/fisiologia , Animais , Western Blotting , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/análise , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular/genética , Células Cultivadas , Condrócitos/química , Colágeno Tipo II/análise , Colágeno Tipo II/genética , Colágeno Tipo II/fisiologia , Colágeno Tipo X/análise , Colágeno Tipo X/genética , Colágeno Tipo X/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/análise , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Fibroblastos/química , Fibroblastos/patologia , Fibroblastos/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Lâmina de Crescimento/química , Lâmina de Crescimento/patologia , Lâmina de Crescimento/fisiopatologia , Proteínas Hedgehog , Proteínas de Grupo de Alta Mobilidade/análise , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/fisiologia , Hipertrofia/patologia , Hipertrofia/fisiopatologia , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/análise , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Osteogênese/genética , Osteogênese/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9 , Transdução de Sinais/genética , Transativadores/análise , Transativadores/genética , Transativadores/fisiologia , Fatores de Transcrição/análiseRESUMO
Both activating and null mutations of proteins required for canonical WNT signaling have revealed the importance of this pathway for normal skeletal development. However, tissue-specific transcriptional mechanisms through which WNT signaling promotes the differentiation of bone-forming cells have yet to be identified. Here, we address the hypothesis that canonical WNT signaling and the bone-related transcription factor RUNX2/CBFA1/AML3 are functionally linked components of a pathway required for the onset of osteoblast differentiation. Our findings show that, in bone of the SFRP1 (secreted frizzled-related protein-1)-null mouse, which exhibits activated WNT signaling and a high bone mass phenotype, there is a significant increase in expression of T-cell factor (TCF)-1, Runx2, and the RUNX2 target gene osteocalcin. We demonstrate by mutational analysis that a functional TCF regulatory element responsive to canonical WNT signaling resides in the promoter of the Runx2 gene (-97 to -93). By chromatin immunoprecipitation, recruitment of beta-catenin and TCF1 to the endogenous Runx2 gene is shown. Coexpression of TCF1 with canonical WNT proteins resulted in a 2-5-fold activation of Runx2 promoter activity and a 7-8-fold induction of endogenous mRNA in mouse pluripotent mesenchymal and osteoprogenitor cells. This enhancement was abrogated by SFRP1. Taken together, our results provide evidence for direct regulation of Runx2 by canonical WNT signaling and suggest that Runx2 is a target of beta-catenin/TCF1 for the stimulation of bone formation. We propose that WNT/TCF1 signaling, like bone morphogenetic protein/transforming growth factor-beta signaling, activates Runx2 gene expression in mesenchymal cells for the control of osteoblast differentiation and skeletal development.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Osteogênese/genética , Transdução de Sinais , Animais , Diferenciação Celular , Células Cultivadas , Imunoprecipitação da Cromatina , Fibroblastos/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Osteoblastos/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismoRESUMO
Arabidopsis PsbQ, encoding a 16 kDa protein of the oxygen-evolving complex, is regulated by light and is expressed preferentially in leaf tissues. To analyze the components required for light-regulated and organ-specific expression of PsbQA, several promoter constructs were generated and expressed in tobacco. The 2.2 kb promoter could confer organ-specific expression of the reporter gene, whereas regulatory elements for light-dependent induction could not be located within this promoter and the transcribed region extending up to a second exon, represented by a genomic fragment encompassing the gene. The genomic fragment representing the transcribed region, however, could confer light regulation even on a constitutive promoter, as observed by steady-state mRNA analysis in T0 and T1 tobacco plants. The results obtained have led to the conclusion that regulatory elements for organ-specificity mainly reside in the promoter region whereas the transcribed region of the gene has an important role in light regulation.
Assuntos
Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Luz , Nicotiana/genética , Complexo de Proteína do Fotossistema II/genética , Regiões Promotoras Genéticas/genética , Regiões 5' não Traduzidas/genética , Northern Blotting , Caulimovirus/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter/genética , Glucuronidase/análise , Especificidade de Órgãos , Fotossíntese/genética , Plantas Geneticamente Modificadas/genética , RNA Mensageiro/análise , Sequências Reguladoras de Ácido Nucleico/genética , Nicotiana/metabolismo , Transcrição GênicaRESUMO
Previous studies have associated activation of canonical Wnt signaling in osteoblasts with elevated bone formation. Here we report that deletion of the murine Wnt antagonist, secreted frizzled-related protein (sFRP)-1, prolongs and enhances trabecular bone accrual in adult animals. sFRP-1 mRNA was expressed in bones and other tissues of +/+ mice but was not observed in -/- animals. Despite its broad tissue distribution, ablation of sFRP-1 did not affect blood and urine chemistries, most nonskeletal organs, or cortical bone. However, sFRP-1-/- mice exhibited increased trabecular bone mineral density, volume, and mineral apposition rate when compared with +/+ controls. The heightened trabecular bone mass of sFRP-1-/- mice was observed in adult animals between the ages of 13-52 wk, occurred in multiple skeletal sites, and was seen in both sexes. Mechanistically, loss of sFRP-1 reduced osteoblast and osteocyte apoptosis in vivo. In addition, deletion of sFRP-1 inhibited osteoblast lineage cell apoptosis while enhancing the proliferation and differentiation of these cells in vitro. Ablation of sFRP-1 also increased osteoclastogenesis in vitro, although changes in bone resorption were not observed in intact animals in vivo. Our findings demonstrate that deletion of sFRP-1 preferentially activates Wnt signaling in osteoblasts, leading to enhanced trabecular bone formation in adults.