Jing Luan

Exosomes derived from mineralizing osteoblasts promote ST2 cell osteogenic differentiation by alteration of microRNA expression

Mineralizing osteoblasts (MOBs) can release exosomes, although the functional significance remains unclear. In the present study, we demonstrate that exosomes derived from mineralizing pre‐osteoblast MC3T3‐E1 cells can promote bone marrow stromal cell (ST2) differentiation to osteoblasts. We reveal that MOB‐derived exosomes significantly influence miRNA profiles in recipient ST2 cells, and these changes tend to activate the Wnt signaling pathway by inhibiting Axin1 expression and increasing β‐catenin expression. We also suggest that MOB derived‐exosomes partly induce the variation in miRNA expression in recipient ST2 cells by exosomal miRNA transfer. These findings suggest an exosome‐mediated mode of cell‐to‐cell communication in the osteogenic microenvironment, and also indicate the potential of MOB exosomes in bone tissue engineering.

L-carnitine affects osteoblast differentiation in NIH3T3 fibroblasts by the IGF-1/PI3K/Akt signalling pathway.

Fibroblasts in soft tissues are one of the progenitors of ectopic calcification. Our previous experiment found that the serum concentrations of small metabolite L-carnitine (LC) decreased in an ectopic calcification animal model, indicating LC is a potential calcification or mineralization inhibitor. In this study, we investigated the effect of LC on NIH3T3 fibroblast osteoblast differentiation, and explored its possible molecular mechanisms. Two concentrations of LC (10 μM and 100 μM) were added in Pi-induced NIH3T3 fibroblasts, cell proliferation was compared by MTT assays, osteoblast differentiation was evaluated by ALP activity, mineralized nodules formation, calcium deposition, and expressions of the osteogenic marker genes. Our results indicated that 10 μM LC increased the proliferation of NIH3T3 cells, but 100 μM LC slightly inhibited cell proliferation. 100 μM LC inhibits NIH3T3 differentiation as evidenced by decreases in ALP activity, mineralized nodule formation, calcium deposition, and down-regulation of the osteogenic marker genes ALP, Runx2 and OCN, meanwhile 10 μM of LC exerts an opposite effect that promotes NIH3T3 osteogenesis. Mechanistically, 100 μM LC significantly inhibits IGF-1/PI3K/Akt signalling, while 10 μM LC slightly activates this pathway. Our study suggests that a decease in LC level might contribute to the development of ectopic calcification in fibroblasts by affecting IGF-1/PI3K/Akt, and addition of LC may benefit patients with ectopic calcification.

A systematic review of genetic skeletal disorders reported in Chinese biomedical journals between 1978 and 2012

Little information is available on the prevalence, geographic distribution and mutation spectrum of genetic skeletal disorders (GSDs) in China. This study systematically reviewed GSDs as defined in “Nosology and Classification of genetic skeletal disorders (2010 version)” using Chinese biomedical literature published over the past 34 years from 1978 to 2012. In total, 16,099 GSDs have been reported. The most frequently reported disorders were Marfan syndrome, osteogenesis imperfecta, fibrous dysplasia, mucopolysaccharidosis, multiple cartilaginous exostoses, neurofibromatosis type 1 (NF1), osteopetrosis, achondroplasia, enchondromatosis (Ollier), and osteopoikilosis, accounting for 76.5% (12,312 cases) of the total cases. Five groups (group 8, 12, 14, 18, 21) defined by “Nosology and Classification of genetic skeletal disorders” have not been reported in the Chinese biomedical literature. Gene mutation testing was performed in only a minor portion of the 16,099 cases of GSDs (187 cases, 1.16%). In total, 37 genes for 41 different GSDs were reported in Chinese biomedical literature, including 43 novel mutations. This review revealed a significant imbalance in rare disease identification in terms of geographic regions and hospital levels, suggesting the need to create a national multi-level network to meet the specific challenge of care for rare diseases in China.

Platelet-derived growth factor receptor kinase inhibitor AG-1295 promotes osteoblast differentiation in MC3T3-E1 cells via the Erk pathway.

Previous studies have conflicting views on the effect of platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) signaling on osteogenesis. The current study investigated the effect of PDGF receptor-beta (PDGFR-β) inhibition by AG-1295 on the osteogenic differentiation of the mouse pre-osteoblastic cell line MC3T3-E1. Osteogenic differentiation was induced by treatment with β-glycerophosphate, ascorbic acid, and dexamethasone along with or absent AG-1295. Results showed that AG-1295 significantly increased alkaline phosphatase (ALP) activity and enhanced the formation of mineralized nodules in a dose-dependent manner. Furthermore, treatment with AG-1295 resulted in up-regulated mRNA expression of the osteogenic marker genes collagen type I (Col1A), runt-related transcription factor 2 (Runx2), osterix (Osx), tissue-nonspecific alkaline phosphatase (Tnap), and osteocalcin (Ocn). Consistent with its effect on osteoblast differentiation, AG-1295 also significantly suppressed the phosphorylation of Erk1/2 in MC3T3-E1 cells. In conclusion, findings suggest that blocking the PDGFR-β pathway with AG1295 markedly promotes osteoblast differentiation and matrix mineralization in mouse osteoblastic MC3T3-E1 cells and that the Erk1/2 pathway might participate in this process.

MiR-5100 promotes osteogenic differentiation by targeting Tob2

MicroRNAs have emerged as pivotal regulators in various physiological and pathological processes, including osteogenesis. Here we discuss the contribution of miR-5100 to osteoblast differentiation and mineralization. We found that miR-5100 was upregulated during osteoblast differentiation in ST2 and MC3T3-E1 cells. Next, we verified that miR-5100 can promote osteogenic differentiation with gain-of-function and loss-of-function experiments. Target prediction analysis and experimental validation demonstrated that Tob2, which acts as a negative regulator of osteogenesis, was negatively regulated by miR-5100. Furthermore, we confirmed that the important bone-related transcription factor osterix, which can be degraded by binding to Tob2, was influenced by miR-5100 during osteoblast differentiation. Collectively, our results revealed a new molecular mechanism that fine-tunes osteoblast differentiation through miR-5100/Tob2/osterix networks.

Epidemiology, diagnosis, and treatment of Wilson's disease

Wilsons disease (WD) is an autosomal recessive disease caused by a mutation of the ATP7B gene, resulting in abnormal copper metabolism. The major clinical features of WD include liver disease, neurological disorders, K-F rings, and osteoporosis. The prevalence of WD in China is higher than that in Western countries. Early diagnosis and lifelong treatment will lead to better outcomes. Drugs such as sodium dimercaptosuccinate (Na-DMPS), Zn, and Gandou Decoction can be used to treat WD. Some studies have shown that the combination of traditional Chinese medicine and Western medicine is the best approach to treating WD. In order to identify better treatments, this article describes the specific clinical symptoms of Wilsons disease, its diagnosis, and treatment options.

Identification of the receptor tyrosine kinases (RTKs)-oriented functional targets of miR-206 by an antibody-based protein array

This study demonstrated the feasibility and benefit of an antibody‐based experimental approach to identify microRNA functional targets from hundreds of predicted genes using miR‐206 as an example. Using a receptor tyrosine kinase (RTK) antibody array, we identified 7 phosphorylated RTKs that were significantly differentially regulated after miR‐206‐mimic transfection. We then focused on MET, the most varied RTK, and bioinformatically constructed a MET‐centred network using computationally predicted miR‐206 targets. Within this network, we analyzed two validated targets, PAX3 and SNX2, and one candidate target, EIF4E, may account for the inhibitory effect of miR‐206 on MET phosphorylation. Luciferase and Western‐blot assays indicated that EIF4E was a direct target of miR‐206. This concept may also be applicable for other microRNAs and other antibody array platforms.

A comparative proteomics study on matrix vesicles of osteoblast- like Saos-2 and U2-OS cells

Matrix vesicles (MVs) play an important role in the initial stage of the process of bone mineralization, and are involved in multiple rare skeletal diseases with pathological mineralization or calcification. The aim of the study was to compare the proteomic profiling of osteoblast-like cells with and without mineralization ability (Saos-2 and U2-OS), and to identify novel mineralization-associated MV proteins. MVs were extracted using ExoQuick solution from mineralization-induced Saos-2 and U2-OS cells, and then were validated by transmission electron microscopy. A label-free quantitative proteomic method was used to compare the protein profiling of MVs from Saos-2 and U2-OS cells. Western-blots were used to confirm the expression of MVs proteins identified in proteomic studies. In our proteomic studies, we identified that 89 mineralization-related proteins were significantly up-regulated in Saos-2 MVs compared with U2-OS MVs. We further validated that two MVs proteins, protein kinase C α and ras-related protein Ral-A, were up-regulated in MVs of Saos-2 cells compared to those of U2-OS cells under mineralization-induction. Our findings suggest that protein kinase C α and ras-related protein Ral-A might be involved in bone mineralization as MVs components.

Fibrodysplasia ossificans progressiva: Basic understanding and experimental models

Fibrodysplasia ossificans progressive (FOP) is an extremely rare autosomal dominant disorder characterized by congenital malformations of the great toes and progressive heterotopic ossification that can induce a disabling second skeleton. Spontaneously occurring flare-ups can cause inflammatory soft tissue to swell, followed by progressive and disabling heterotopic endochondral ossification. FOP is very rare, with an estimated incidence of one case per two million individuals. There is no definitive treatment for FOP, but the longevity of patients with FOP can be extended by early diagnosis and appropriate prevention of flares-up. Some promising treatment strategies and targets have recently been reported. The current review describes the classical phenotype and genotype of FOP, useful methods of diagnosing the condition, therapeutic approaches and commonly used drugs, and experimental models used to study this disease.

Establishment of a human trisomy 18 induced pluripotent stem cell line from amniotic fluid cells

Trisomy 18 (18T) is the second most common autosomal trisomy syndrome in humans, but the detailed mechanism of its pathology remains unclear due to the lack of appropriate models of this disease. To resolve this problem, the current study reprogrammed human 18T amniotic fluid cells (AFCs) into an induced pluripotent stem cell (iPSC) line by introducing integration-free episomal vectors carrying pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1. The pluripotency of 18T-iPSCs was subsequently validated by alkaline phosphatase staining, detection of iPSC biomarkers using real-time PCR and flow cytometry, detection of embryoid body (EB) formation, and detection of in vivo teratoma formation. Moreover, this study also investigated the transcriptomic profiles of 18T-iPSCs using RNA sequencing, and several gene clusters associated with the clinical manifestations of 18T were identified. In summary, the generated induced pluripotent stem cells line has typical pluripotency characteristics and can provide a useful tool with which to understand the development of 18T.