DNA methylation analysis identifies key transcription factors involved in mesenchymal stem cell osteogenic differentiation

BACKGROUND: Knowledge about regulating transcription factors (TFs) for osteoblastogenesis from mesenchymal stem cells (MSCs) is limited. Therefore, we investigated the relationship between genomic regions subject to DNA-methylation changes during osteoblastogenesis and the TFs known to directly interact with these regulatory regions. RESULTS: The genome-wide DNA-methylation signature of MSCs differentiated to osteoblasts and adipocytes was determined using the Illumina HumanMethylation450 BeadChip array. During adipogenesis no CpGs passed our test for significant methylation changes. Oppositely, during osteoblastogenesis we identified 2462 differently significantly methylated CpGs (adj. p < 0.05). These resided outside of CpGs islands and were significantly enriched in enhancer regions. We confirmed the correlation between DNA-methylation and gene expression. Accordingly, we developed a bioinformatic tool to analyse differentially methylated regions and the TFs interacting with them. By overlaying our osteoblastogenesis differentially methylated regions with ENCODE TF ChIP-seq data we obtained a set of candidate TFs associated to DNA-methylation changes. Among them, ZEB1 TF was highly related with DNA-methylation. Using RNA interference, we confirmed that ZEB1, and ZEB2, played a key role in adipogenesis and osteoblastogenesis processes. For clinical relevance, ZEB1 mRNA expression in human bone samples was evaluated. This expression positively correlated with weight, body mass index, and PPARγ expression. CONCLUSIONS: In this work we describe an osteoblastogenesis-associated DNA-methylation profile and, using these data, validate a novel computational tool to identify key TFs associated to age-related disease processes. By means of this tool we identified and confirmed ZEB TFs as mediators involved in the MSCs differentiation to osteoblasts and adipocytes, and obesity-related bone adiposity.

Micro-ARNs y células óseas / Micro RNAs and bone cells

Los micro-ARNs (miARNs) son pequeñas moléculas de ARN no codificante (de aproximadamente 15-25 nucleótidos), que regulan la expresión de genes involucrados en numerosas funciones biológicas, a través de la inhibición o degradación de un ARN mensajero diana. La homeostasis ósea se mantiene por el balance entre la formación osteoblástica y la resorción osteoclástica. La sobreexpresión o inhibición de miARNs específicos afecta la proliferación, diferenciación y actividad de osteoblastos, osteocitos y osteoclastos. Estas acciones son llevadas a cabo modulando la expresión de distintos factores transcripcionales y moléculas de señalización de las vías esenciales para la osteoblastogénesis u osteoclastogénesis. Estos efectos modifican el balance entre la formación y la resorción, determinando cambios en la homeostasis ósea. Esta revisión enumera una serie de miARNs que participan en la homeostasis ósea. Profundizando en el conocimiento de los mecanismos por medio de los cuales los miARNs actúan sobre el hueso, podrían revelarse nuevos usos potenciales futuros, entre los que se encuentran su utilidad como nuevos biomarcadores óseos o como agentes terapéuticos para el tratamiento de trastornos metabólicos óseos, pérdida de masa ósea o enfermedades óseas. (AU)

MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules (of approximately 15­25 nucleotides), which regulate the expression of genes controlling numerous biological functions, through the inhibition or degradation of the target messenger RNA. Bone homeostasis is maintained by a balance between osteoblastic bone formation and osteoclastic bone resorption. The overexpression or inhibition of specific miRNAs affects cell proliferation, differentiation and activity of osteoblast, osteocytes and osteoclast. This action is done by modulating the expression of different transcription factors and signaling molecules of the most relevant pathways of osteoblastogenesis or osteoclastogenesis. This effect is able to modify the balance between bone formation and resorption, determining changes in bone homeostasis. The present review is an overview of a series of miRNAs involved in bone homeostasis. An in depth knowledge of the mechanisms by which miRNAs act on bone may reveal potential uses in the future as new bone biomarkers or therapeutic agents for treating metabolic bone disorders, bone loss and bone diseases. (AU)

SPA0355 prevents ovariectomy-induced bone loss in mice

Estrogen withdrawal in post-menopausal women leads to overactivation of osteoclasts, which contributes to the development of osteoporosis. Inflammatory cytokines are known as one of mechanisms of osteoclast activation after estrogen deficiency. SPA0355 is a thiourea derivative that has been investigated for its antioxidant and anti-inflammatory activities. However, its efficacy in bone resorption has not been previously investigated. The aim of this study was to investigate the impact of SPA0355 on the development of osteoporosis and to explore its mode of action. In vitro experiments showed that SPA0355 inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in primary bone marrow-derived macrophages. This effect appears to be independent of estrogen receptor activation as ICI 180,782 failed to abrogate its effects on osteoclasts. Further signaling studies revealed that SPA0355 suppressed activation of the MAPKs, Akt, and NF-κB pathways. SPA0355 also increased osteoblastic differentiation, as evidenced by its effects on alkaline phosphatase activity and mineralization nodule formation. Intraperitoneal administration of SPA0355 to ovariectomized mice prevented bone loss, as verified by three-dimensional images and bone morphometric parameters derived from µCT analysis. Noticeably, SPA0355 did not show hepatotoxicity and nephrotoxicity and also had little effect on hematological parameters. Taken together, the results indicate that SPA0355 may protect against bone loss in ovariectomized mice by stimulation of osteoblast differentiation and by inhibition of osteoclast resorption. Therefore, SPA0355 is a safe and potential candidate for management of postmenopausal osteoporosis.

A review for function of Wnt protein family in osteocyte / 医学研究生学报

Wnt signaling pathway has a critical role in differentiation, proliferation, supersession and metabolism of osteocyte. Although the principal components are relatively simple, however, the numerous components consisted of Wnt signaling participate in the reaction, including several LRPs ( low density lipoprotein receptor-related protein,LRP) , 19 different Wnt proteins and 19 different FZDs( frizzled receptor, FZD) , and non-canonical Wnt signaling and multiple inhibitors of Wnt signaling.The Wnts linked to mem-brane coreceptor and activated downstream of canonical Wnt signaling or non-canonical Wnt signaling, In addition, different Wnts had different roles in differentiation, bone formation and bone absorption of osteocyte.This review aims to understand the relationship be-tween Wnt signaling and osteocyte metabolism and bone diseases, by analyzing a variety of factors and the process of Wnt protein and Wnt signaling pathway in activity of osteocyte.

Fosfoproteômica e proteômica quantitativa de células mesenquimais durante a diferenciação osteoblástica mediada por BMP2, expressão e purificação de diferentes tipos de proteínas morfogenéticas ósseas / Quantitative phosphoproteomics and proteomics of mesenchymal stem cells during BMP2-mediated osteoblastic differentiation, expression and purification of different types of bone morphogenetic proteins

As fraturas e perdas ósseas representam altos riscos para o Sistema público de Saúde (SUS), além de afetar a qualidade de vida do paciente, portanto é necessário o entendimento das bases moleculares que envolvem os mecanismos de reparo ósseo. Citocinas secretadas por células do sistema imune presentes no local da inflamação, como as IL-6, IL-10 e TNFα atuam como fatores quimiotáticos para células mesenquimais, que proliferam e se diferenciam em osteoblastos pela ação autócrina e parácrina de Proteínas Morfogenéticas Ósseas (BMPs), principalmente a BMP2. Embora seja conhecido que a ação de BMP2 ocorra através de sua ligação nos receptores ActRI/BMPR, que ativam proteínas SMADS 1/5/8 efetoras, pouco se sabe sobre os mecanismos intracelulares que participam do processo de diferenciação osteoblástico. Neste estudo propôs-se analisar as diferenças no conteúdo de proteínas totais e de proteínas fosforiladas em células mesenquimais de pele induzidas à osteogênese pelo tratamento com BMP2 por diferentes períodos de tempo, utilizando-se de Isótopos Estáveis de Dimetila acoplado ao LC/MS. A partir de 150µg de material inicial, foi possível identificar 2.264 proteínas, as quais foram quantificadas nos diferentes pontos de indução, sendo que 235 são fosforiladas. Análise de motivos de quinases mostrou que diversos substratos possuem sítios fosforilados correspondentes àqueles dos motivos de fosforilação das quinases Casein Kinase, p38, CDK e JNK. A análise da ontologia gênica mostrou um aumento de processos biológicos relacionados com sinalização e diferenciação após a primeira hora de indução com rhBMP2. Além disso, proteínas envolvidas com o rearranjo do citoesqueleto e com vias de sinalização Wnt e Ras foram encontradas como tendo fosforilação diferencial durante todos os períodos estudados. Os dados revelaram novos substratos intracelulares que são fosforilados nos primeiros momentos do comprometimento com a diferenciação osteoblástica mediada pelo tratamento com rhBMP2 em células mesenquimais derivadas da pele. Além disso, clones celulares que superexpressam as proteínas recombinantes humanas BMP2 e BMP4 foram gerados, e sua atividade verificada in vitro. Paralelamente, a rhBMP7, obtida anteriormente, foi purificada por cromatografia de afinidade utilizando-se uma coluna de Heparina-Sepharose, que foi posteriormente utilizada para ensaios in vitro e in vivo, nos quais se mostrou capaz de gerar osteoblastos e tecido ósseo, respectivamente, o que abre novas possibilidades para o uso destas proteínas como biofármacos no Brasil

Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated human skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. From 150 µg of starting material, 2,264 proteins containing two or more peptides were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during commitment to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells. Cell clones overexpressing the human BMP 2 and 4 recombinant proteins were also generated, and their biological activity was confirmed in vitro. In parallel, chromatography-affinity purified rhBMP7, obtained using heparin-Sepharose columns, was used for in vivo and in vitro assays to evaluate the ability of this purified protein to generate osteoblasts and bone tissue, respectively, opening new avenues for the use of these proteins as biopharmaceuticals in Brazil

Petroleum ether extract of Cissus quadrangularis (Linn. ) enhances bone marrow mesenchymal stem cell proliferation and facilitates osteoblastogenesis

OBJECTIVE: To evaluate the effects of the petroleum ether extract of Cissus quadrangularis on the proliferation rate of bone marrow mesenchymal stem cells, the differentiation of marrow mesenchymal stem cells into osteoblasts (osteoblastogenesis) and extracellular matrix calcification. This study also aimed to determine the additive effect of osteogenic media and Cissus quadrangularis on proliferation, differentiation and calcification. METHODS: MSCs were cultured in media with or without Cissus quadrangularis for 4 weeks and were then stained for alkaline phosphatase. Extracellular matrix calcification was confirmed by Von Kossa staining. marrow mesenchymal stem cells cultures in control media and osteogenic media supplemented with Cissus quadrangularis extract (100, 200, 300 µg/mL) were also subjected to a cell proliferation assay (MTT). RESULTS: Treatment with 100, 200 or 300 µg/mL petroleum ether extract of Cissus quadrangularis enhanced the differentiation of marrow mesenchymal stem cells into ALP-positive osteoblasts and increased extracellular matrix calcification. Treatment with 300 µg/mL petroleum ether extract of Cissus quadrangularis also enhanced the proliferation rate of the marrow mesenchymal stem cells. Cells grown in osteogenic media containing Cissus quadrangularis exhibited higher proliferation, differentiation and calcification rates than did control cells. CONCLUSION: The results suggest that Cissus quadrangularis stimulates osteoblastogenesis and can be used as preventive/alternative natural medicine for bone diseases such as osteoporosis.