Regulation of osteosarcoma and stem cell-related signaling pathways / 中国组织工程研究

BACKGROUND: Osteosarcoma is the most common primary bone-derived malignancy in children and adolescents. The prognosis of osteosarcoma patients has hardly improved over the past 30 years. It is urgent to develop new strategies and innovative therapies to further improve the survival rate of osteosarcoma patients. OBJECTIVE: To explain the latest advances in molecular mechanisms and signaling networks that contribute to the progression and metastasis of osteosarcoma, and provide a theoretical basis for the treatment of osteosarcoma. METHODS: The “osteosarcoma, signaling pathway, prognosis, metastasis, drug resistance, Notch, Wnt/β-catenin, Hedgehog, PI3K/Akt/mTOR, BMPs” were as keywords to search the articles in the CNKI and PubMed databases published from February 1999 to February 2019. Totally 586 related articles were retrieved. After reading the title and abstract, irrelevant articles were excluded based on exclusion criteria, and finally 40 eligible articles were included for review. RESULTS AND CONCLUSION: Signaling pathways are critical in the regulation of osteosarcoma formation and proliferation. Studies have found that Notch, Wnt/β-catenin, Hedgehog, PI3K/Akt/mTOR and BMP signaling pathways are expressed in osteosarcoma and stem cells, which have an important impact on the occurrence and development of osteosarcoma.

Effect of aqueous extract from Araliae Echinocaulis Radicis et Cortex containing serum on TGF-β/BMPs signaling pathway in primary osteoblasts / 药物评价研究

Objective To study the effect of aqueous extract from Araliae Echinocaulis Radicis et Cortex (AEAE) on expression of TGF-β/BMPs signaling pathway in osteoblasts.Methods The fracture model of rat was established and randomly divided into model group,AEAE low,middle and high dose (3.6,1.8,0.9 g/kg) group.After ig administration for 7 d,the blood was taken from abdominal aorta and the serum was separated.The rats primary osteoblasts were cultured and qualified after alkaline phosphatase staining and alizarin red staining.The osteoblasts were cultured continuously for 48 h in animal serum of the corresponding group.The contents of BMP-2,TGF-β,Smad-1 and Smad-2 were detected by qRT-PCR and Western blotting method.Results Compared with model group,the mRNA level ofBMP-2,TGF-β and Smad-1 in AEAE low,middle and high dose group,Smad-2 mRNA level in middle and high dose group,and the protein level of BMP-2,TGF-β,Smad-1 and Smad-2 in low,middle and high dose groupwere significantly higher than those in model group (P＜0.05 and 0.01).Conclusion AEAE can promote the proliferation ofosteoblasts by up-regulating the expression of BMP-2,TGF-β,Smad-1 and Smad-2 in TGF-β/BMPs signaling pathway.

Effects of GDF-9 and FSH on mRNA expression for FSH-R, GDF-9 and BMPs in in vitro cultured goat preantral follicles

The aim of the present study was to determine the role of GDF-9 and/or FSH on the growth and mRNA expression for FSH-R, GDF-9, and BMPs in goat secondary follicles after culture in vitro. Goat secondary follicles (~200µm) were isolated and cultured for six days in minimum essential medium (MEM) supplemented with GDF-9 (200 ng/mL), FSH (50 ng/mL) or both. At the beginning and end of culture, the follicular diameter was evaluated and compared. The levels of mRNA for GDF-9, FSH-R and BMPs -2, -4, -6, -7 and -15 in cultured follicles were quantified by real time PCR. The results showed that a significant increase of follicle diameter after six days when compared to day 0, but the presence of GDF-9 and FSH did not influence the follicular growth in comparison with those cultured in MEM. Real time PCR showed that GDF-9 down-regulated the levels of mRNA for BMPs -2 and -15, while FSH either alone or in combination with GDF-9 did not affect the expression of GDF-9, FSH-R and BMPs. In conclusion, GDF-9 reduced the expression of BMP-2 and -15 in caprine preantral follicles after their culture, but FSH either alone or in association with GDF-9 did not control the expression of GDF-9, FSH-R and BMPs.

Study on the role of COX-2 in BMP9 induced osteogenic differentiation in mesenchymal stem cells / 中国药理学通报

Aim To investigate the role of COX-2 in BMP9 induced osteogenic differentiation,and the pos-sible mechanism underlying this function of COX-2. Methods We introduced real-time PCR, Western blot, and immunocytochemical staing to detect the effect of BMP9 on COX-2 expression.We employed chemiluminescence technique to assay ALP activities, RT-PCR to detect the expression of Smad6 and Smad7 , and Western blot to measure the expression of Runx2, Dlx-5,total Smad1/5/8,and phosphorylated Smad1/5/8.Finally,BMPR-Smad luciferase reporter assay was applied to measure the activation of BMPs/Smads signaling.Results BMP9 could induce the expression of COX-2 in C3H10T1/2 cells.Either inhibiting enzy-matic activity of COX-2 or knockdown of the expression of COX-2 reduced the BMP9 induced ALP activities in C3H10T1/2 cells,and COX-2 knockdown also inhibited the ectopic bone formation induced by BMP9 in C3H10T1/2 cells.Moreover,COX-2 knockdown inhibi-ted BMPR-Smad reporter activities and the phosphoryl-ation of Smad1/5/8,so did the expression of Smad6 and Smad7 .Conclusion COX-2 may play an impor-tant role in BMP9 induced osteogenic differentiation in MSCs by regulating the BMPs/Smads signaling trans-duction.

BMPs and cancer / 中南大学学报(医学版)

Bone morphogenetic proteins (BMPs) were first studied as growth factors or morphogens of the transforming growth factor-beta super family. These growth molecules, originally associated with bone and cartilage development, are now known to play important roles in morphogenesis and homeostasis in many other tissues. Recently, signiifcant contributions of BMPs, their receptors, and interacting molecules have been linked to carcinogenesis and tumor progression. BMPs can sometimes play a role as a tumor suppressor. This article explains the composition and biological characteristics of BMPs, and investigates their new roles in the pathogenesis of cancer.

Osteogenic molecules for clinical applications: improving the BMP-collagen system

Among the osteogenic growth factors used for bone tissue engineering, bone morphogenetic proteins (BMPs) are the most extensively studied for use in orthopaedic surgery. BMP-2 and BMP-7 have been widely investigated for developing therapeutic strategies and are the only two approved for use in several clinical applications. Due to the chemical and biological characteristics of these molecules, their authorised uses are always in combination with a carrier based on collagen type I. Although the use of these growth factors is considered safe in the short term, the very high doses needed to obtain significant osteoinduction make these treatments expensive and their long-term safety uncertain, since they are highly pleiotropic and have the capacity to induce ectopic ossification in the surrounding tissues. Therefore it is necessary to improve the currently used BMP-collagen system in terms of efficiency, biosecurity and costs. There are several strategies to increase the clinical effectiveness of these treatments. In this review we summarize the most promising results and our related work focused on this field through two different approaches: i) the development of recombinant BMPs with additional features, and ii) complementing these systems with other growth factors or molecules to enhance or accelerate osteogenesis.

Inhibition of Testudinis Carapax et Plastrum extracts targeting BMP4 pathway on PC12 cell apoptosis / 中草药

Objective: To observe the protection of Testudinis Carapax et Plastrum extracts (TCPE) on serum starvation-induced PC12 cell apoptosis and explore its mechanism. Methods: The PC12 apoptosis model was established by serum starvation for 3 d. The cells were randomly divided into four groups: control group, model group, low-dose and high-dose (3 and 30 μg/mL) TCPE groups. In the three days of the treatment, cell absorbance was determined by MTT, ratio of cell apoptosis was examined by Annexin V/PI double stain flow cytometry (FCM), Caspase-3, BMP4, BMPR-IA, and p-Smad1/5/8 signaling molecular expression were detected by Western blotting, and the anti-apoptotic effect of TCPE was observed after blocking BMPs signal pathway. Semi-quantitative analysis of bands was carried out by Bio-Rad Quantity One gel analysis system. Results: MTT and FCM analyses demonstrated that TCPE could increase PC12 cell viability and decrease their apoptotic ratios in a dose dependent manner. Western blotting results showed that TCPE could decrease Caspase-3 expression, promote the expression of BMP4, BMPR-IA, and p-Smad1/5/8. There was statistically significant difference between TCPE (3 and 30 μg/mL) groups and model group (P<0.05, P<0.01) in all above results. While TCPE had no effect on the expression of BMP2, BMP7, and BMPR-II. BMPR-IB hadn't been detected. The anti-apoptotic activity was partially mitigated by neutralizing BMP4 antibody. Conclusion: TCPE has the capacity to inhibit the apoptosis of PC12 induced by serum starvation in a dose dependent manner and its mechanism may be associated with partially activating and up-regulating the expression of BMP4 signaling pathway.

Análise dos genes diferencialmente expressos durante a osteodiferenciação induzida por proteínas morfogenéticas de osso (BMP2 e BMP7) em células C2C12 e super-expressão de rhBMP2 e rhBMP7 em células de mamíferos / Analysis of differentially expressed genes during osteodifferentiation induced by bone morphogenetic proteins (BMP2 e BMP7) of C2C12 cells and overexpression of rhBMP2 and rhBMP7 in mammalian cells

As BMPs (Bone Morphogenetic Proteins) são membros da superfamília de proteínas TGF-β (Transforming Growth Factor β ), regulam o crescimento e diferenciação de vários tipos celulares em diversos tecidos, e algumas delas desempenham um papel crítico na diferenciação de células de origem mesenquimal em osteoblastos. Particularmente, rhBMP2 e rhBMP7, promovem osteoindução tanto /"in vitro/" como /"in vivo,/" sendo, ambas as proteínas utilizadas terapeuticamente em Ortopedia/Odontologia para reparo ósseo. A expressão diferencial de genes durante a osteodiferenciação de células C2C12 induzida por rhBMP2 e rhBMP7, foi analisada através de microarranjos de DNA, selecionando 31 genes, dos quais 24 foram validados por qPCR, 13 dos quais são relacionados à transcrição, quatro associados a algumas vias de sinalização celular e sete associados à matriz extracelular. Análise funcional destes genes permitirá conhecer, com maiores detalhes, os eventos moleculares que ocorrem durante a diferenciação osteoblástica de células C2C12 induzida por rhBMPs. Em paralelo, foi perseguida a super-expressão de rhBMP2 e rhBMP7 em células HEK293T, demonstrando-se a atividade de rhBMP7, induzindo osteodiferenciação /"in vitro/" e formação de osso /"in vivo/", demonstrando a viabilidade do objetivo de se produzir estas proteínas para futura aplicação como biofármacos no Brasil.

The BMPs (Bone Morphogenetic Proteins) are members of the TGF-β (Transforming Growth Factor β) superfamily of proteins, regulate growth and differentiation of various cell types in various tissues, and some play a critical role in differentiation of mesenchymal cells into osteoblasts. Particularly, rhBMP2 and rhBMP7, promote osteoinduction /"in vitro/" and /"in vivo/" and both proteins are used therapeutically in Orthopedics and Dentistry. The differential expression of genes during osteodifferentiation induced by rhBMP2 and rhBMP7 in C2C12 cells was analyzed through DNA microarrays, allowing the selection of 31 genes, of which 24 were validated by qPCR, 13 of which are related to transcription, four associated with cell signaling pathways and seven are associated with the extracellular matrix. Subsequent functional analysis of these genes should reveal more details on the molecular events which take place during C2C12 cells osteoblastic differentiation induced by rhBMPs In paralel, rhBMPs 2 and 7 were overexpressed in HEK293T cells and BMP7 activity to induce osteodifferentiation /"in vitro/" and bone formation /"in vivo/" was demonstrated, reinforcing the viability of our objective to produce these proteins for future application as biopharmaceuticals in Brazil

The Role of E3 Ubiquitin Ligase Smurfs in BMP and TGF-bata Signaling in Bone Cells / 生物化学与生物物理进展

The ubiquitin-proteasome system is composed by multiple enzymes which are ubiquitously expressed in mammalian cells and plays an essential role in a variety of biological processes. As key members of the protein degradation enzymatic system, the function of E3 ubiquitin ligases has been extensively investigated. BMP and TGF-? are critical molecules that regulate proliferation, differentiation and apoptosis of osteoblasts and chondrocytes through different signaling pathways. Resent findings indicate that the ubiquitin-proteasome system functions as a key regulator in bone cells and the E3 ligase mediates the proteolytic degradation of critical molecules in BMP and TGF-? signaling pathways. Recent progress on studies of HECT domain E3 ligase, Smurf, in osteoblasts and chondrocytes were summarized. The regulatory role of Smurf1 and Smurf2 in BMP and TGF-? signaling and osteoblast and chondrocyte function has been reviewed.

Expression of BMP-2/4 and BMPR-IA in oral squamous cell carainoma / 实用口腔医学杂志

Objective: To study the relationship between bone morphogenetic proteins(BMPs),BMP receptor type IA (BMPR IA) and the oncogenesis of squamous cell carcinoma in oral epithelia. Methods: Expression of BMP 2/4,BMPR IA was immunohistochemistry analyzed in the specimens obtained from buccal mucosa, including 18 cases of normal mucosa(NB), 24 cases of chronic inflammation(NCI) and 58 cases of oral squamous cell carcinoma(SCC). Result: Weak staining of BMP 2/4 and BMPR IA was observed in normal and chronic inflammation samples,while stroug staining was found in 52 out of 58 cases of oral SCC. No significant difference was found among the heighly,intermediately and poorly differentiated SCC groups.Conclusion: BMP 2/4,BMP IA might be involved in oncogenesis and development of squamous cell carcinoma of oral epithelium.