Yaping Ye

miR‑142‑3p promotes osteoblast differentiation by modulating Wnt signaling

Canonical Wnt signaling is critical for the control of osteoblast differentiation in human mesenchymal stem cells. MicroRNAs (miRs) are essential regulators of cell differentiation by post‑transcriptional regulation of target gene expression. The aim of the present study was to investigate the molecular mechanism by which miR‑142‑3p promotes osteoblastic differentiation using the human fetal osteoblastic 1.19 (hFOB1.19), real-time PCR and western blot analysis. Results showed an increased expression of miR‑142‑3p during osteoblast differentiation in the mesenchymal precursor cell line, hFOB1.19. In addition, the ectopic over-expression of miR‑142‑3p promoted hFOB1.19 differentiation, whereas the inhibition of miR‑142‑3p repressed differentiation. The expression of miR‑142‑3p was positively correlated with β‑catenin, an important protein in Wnt signaling. The adenomatous polyposis coli (APC) gene was a direct target of miR‑142‑3p, whereby miR‑142‑3p promoted Wnt signaling through inhibition of APC, leading to accumulation and nuclear translocation of β‑catenin. Therefore, miR‑142‑3p may be an essential mediator of osteoblast differentiation and a new therapeutic strategy for osteogenesis disorders.

Inhibitory effects of high glucose/insulin environment on osteoclast formation and resorption in vitro

Patients with type 2 diabetes mellitus (T2DM) exhibit hyperglycemia and hyperinsulinemia and increased risk of fracture at early stage, but they were found to have normal or even enhanced bone mineral density (BMD). This study was aimed to examine the molecular mechanisms governing changes in bone structure and integrity under both hyperglycemic and hyperinsulinemic conditions. Monocytes were isolated from the bone marrow of the C57BL/6 mice, induced to differentiate into osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) and exposed to high glucose (33.6 mmol/L), high insulin (1 μmol/L), or a combination of high glucose/high insulin (33.6 mmol/L glucose and 1 μmol/L insulin). Cells cultured in α-MEM alone served as control. After four days of incubation, the cells were harvested and stained for tartrate resistant acid phosphatase (TRAP). Osteoclast-related genes including RANK, cathepsin K and TRAP were determined by using real-time PCR. The resorptive activity of osteoclasts was measured by using a pit formation assay. Osteoclasts that were derived from monocytes were of multinucleated nature and positive for TRAP, a characteristic marker of osteoclasts. Cell counting showed that the number of osteoclasts was much less in high glucose and high glucose/high insulin groups than in normal glucose and high insulin groups. The expression levels of RANK and cathepsin K were significantly decreased in high glucose, high insulin and high glucose/high insulin groups as compared with normal glucose group, and the TRAP activity was substantially inhibited in high glucose environment. The pit formation assay revealed that the resorptive activity of osteoclasts was obviously decreased in high glucose group and high glucose/high insulin group as compared with normal group. It was concluded that osteoclastogenesis is suppressed under hyperglycemic and hyperinsulinemic conditions, suggesting a disruption of the bone metabolism in diabetic patients.SummaryPatients with type 2 diabetes mellitus (T2DM) exhibit hyperglycemia and hyperinsulinemia and increased risk of fracture at early stage, but they were found to have normal or even enhanced bone mineral density (BMD). This study was aimed to examine the molecular mechanisms governing changes in bone structure and integrity under both hyperglycemic and hyperinsulinemic conditions. Monocytes were isolated from the bone marrow of the C57BL/6 mice, induced to differentiate into osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) and exposed to high glucose (33.6 mmol/L), high insulin (1 μmol/L), or a combination of high glucose/high insulin (33.6 mmol/L glucose and 1 μmol/L insulin). Cells cultured in α-MEM alone served as control. After four days of incubation, the cells were harvested and stained for tartrate resistant acid phosphatase (TRAP). Osteoclast-related genes including RANK, cathepsin K and TRAP were determined by using real-time PCR. The resorptive activity of osteoclasts was measured by using a pit formation assay. Osteoclasts that were derived from monocytes were of multinucleated nature and positive for TRAP, a characteristic marker of osteoclasts. Cell counting showed that the number of osteoclasts was much less in high glucose and high glucose/high insulin groups than in normal glucose and high insulin groups. The expression levels of RANK and cathepsin K were significantly decreased in high glucose, high insulin and high glucose/high insulin groups as compared with normal glucose group, and the TRAP activity was substantially inhibited in high glucose environment. The pit formation assay revealed that the resorptive activity of osteoclasts was obviously decreased in high glucose group and high glucose/high insulin group as compared with normal group. It was concluded that osteoclastogenesis is suppressed under hyperglycemic and hyperinsulinemic conditions, suggesting a disruption of the bone metabolism in diabetic patients.

Dose-dependent inhibitory effects of zoledronic acid on osteoblast viability and function in vitro

Zoledronic acid (ZA), which is one of the most potent and efficacious bisphosphonates, has been commonly used in clinical practice for the treatment of various bone disorders. The extensive use of ZA has been associated with increasing occurrence of jaw complications, now known as bisphosphonate-associated osteonecrosis of the jaw (BRONJ). However, the mechanism underlying BRONJ remains to be fully elucidated. The aim of the present study was to investigate the effects of different concentrations of ZA on the MC3T3-E1 murine preosteoblast cell line cells and examine the possible pathogenesis of BRONJ. In the present study, the effect of ZA on the viability, apoptosis, differentiation and maturation of MC3T3-E1 cells, as well as its relevant molecular mechanism, were examined The results of a Cell Counting Kit 8 assay, a flow cytometric Annexin-V/propidium iodide assay and western blot analysis demonstrated that ZA exhibited a significant inhibition of cell viability and induction of apoptosis at concentrations >10 µM. Subsequently, the effect of ZA on cell differentiation at concentrations <1 µM were investigated. In this condition, ZA inhibited bone nodule formation and decreased the activity of alkaline phosphatase. The results of reverse transcription-quantitative polymerase chain reaction and western blot analyses indicated that ZA downregulated the expression levels of the marker genes and proteins associated with osteogenic differentiation. Further investigation revealed that the suppression of differentiation by ZA was associated with decreased expression of bone morphogenetic protein-2 (BMP-2) and downregulation of the phosphorylation levels in the downstream extracellular signal-regulated kinase 1/2 and p38 pathways. These adverse effects of ZA were observed to be concentration-dependent. The results from the present study suggested that ZA at higher concentrations induces cytotoxicity towards osteoblasts, and ZA at lower concentrations suppresses osteoblast differentiation by downregulation of BMP-2. These results assist in further understanding the mechanisms of BRONJ.

Glycosaminoglycan chains of biglycan promote bone morphogenetic protein-4-induced osteoblast differentiation

Biglycan (BGN) has been reported to promote bone morphogenetic protein-4 (BMP-4) stimulated osteoblastic differentiation. However, the underlying mechanism has yet to be fully elucidated. The glycosaminoglycan (GAG) chains of BGN have a variety of biological functions. In the present study, we explored the potential role of the GAG chains of BGN in promoting BMP-4-induced osteoblast differentiation. BGN knockout (KO) murine calvarial cells were transfected with adenovirus overexpressing wild-type BGN (Adv-BGN), adenovirus expressing GAG-mutant BGN (Adv-BGNm) and adenovirus without BGN (Adv-Emp). Transfected cells were treated with or without BMP-4. Subsequently, BMP-4 signaling and function were assessed by evaluating the expression of the osteoblast differentiation-related proteins, Smad1/5/8 phosphorylation and alkaline phosphatase (ALP) activity. Furthermore, the binding specificity of the transfected cells to BMP-4 was also investigated using immunofluorescence staining. Our study demonstrated that a mutant BGN lacking GAG chains decreased BGN-assisted BMP-4 signaling and osteoblast differentiation and that the expression of this mutant BGN in biglycan knockout (BGN‑KO) calvarial osteoblasts could not rescue its differentiation deficiency as efficiently as wild-type (WT) BGN. These results strongly suggest that the GAG chains of BGN promote BGN-assisted BMP-4 function.

Bone morphogenetic proteins induce rabbit bone marrow-derived mesenchyme stem cells to differentiate into osteoblasts via BMP signals pathway.

Abstract Bone marrow mesenchymal stem cells (BMMSCs) are multipotent stem cells that can differentiate into different blastoderm cells in vitro. In this study, BMMSCs in rabbit bone marrow were isolated by density gradient centrifuge separation, purified and expanded in vitro. BMP-2 and FGF 2 were used for differentiation into osteoblasts, and the results demonstrated that bone morphogenetic proteins (BMPs) could affect the differential direction of the BMMSCs. PCR assays indicated that BMP signals pathway played important roles in osteoblasts differentiation of BMMSCs, and the members included BMPRI, Smad 1, Smad 5, Smad 8, Runx 2, collage type I and osteopontin. This study provides a theoretical basis and experimental evidence for the therapeutic application of BMMSCs to the treatment of bone injury.

Comparative study of the osteogenic differentiation capacity of human bone marrow- and human adipose-derived stem cells under cyclic tensile stretch using quantitative analysis

Studies comparing the osteogenic differentiation capacity of human bone marrow-derived stem cells (hBMSCs) and human adipose-derived stem cells (hASCs) cultured in osteogenic differentiation medium have been inconclusive. Apart from chemical stimuli, mechanical stimuli have also been shown to be important in bone tissue engineering, which is referred to as functional bone tissue engineering. hBMSCs and hASCs have been shown to be sensitive to both chemical and mechanical stimuli. In an attempt to find a better seed cell in functional bone tissue engineering, we tried to quantify the osteogenic differentiation capacity of hBMSCs and hASCs under both mechanical and chemical stimuli. In this study, hBMSCs and hASCs were isolated from the same volunteers. Cells were cultured in osteogenic differentiation medium with and without exposure to cyclic tensile stretch (CTS). Quantitative measurement of alkaline phosphatase (ALP) activity revealed that the osteogenic differentiation capacity of hBMSCs was similar to that of hASCs in the early phase of differentiation in the CTS-stimulated groups. Quantitative measurement of mineralization showed that the late-phase osteogenic differentiation capacity of the hBMSCs was superior to that of hASCs in the CTS-stimulated groups. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed 5 and 10 days after cell culture. The results of the RT-PCR revealed that the osteogenic differentiation capacity of hASCs was inferior to that of hBMSCs both in the CTS-stimulated and unstimulated groups. All the results showed that both hBMSCs and hASCs were sensitive to CTS during the osteogenic differentiation process. This study compared the osteogenic differentiation capacity of hBMSCs and hASCs in response to mechanical stimulations and has important implications for the use of stem cells in functional bone tissue engineering and regenerative medicine.

Alterations in enhancer of zeste homolog 2, matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 expression are associated with ex vivo and in vitro bone metastasis in renal cell carcinoma

Renal cell carcinoma (RCC) has a high potential for bone metastasis; however, the molecular mechanisms underlying this metastasis have remained to be elucidated. The present study aimed to explore the expression levels of enhancer of zeste homolog 2 (EZH2), matrix metalloproteinase-2 (MMP2) and tissue inhibitor of metalloproteinase-2 (TIMP2) as determinants of RCC-associated bone metastasis. Their expression was evaluated in a newly generated RCC cell subline that has a high potential for bone metastasis, in tissue specimens from metastasized bone tissues from patients with RCC and in RCC tissues without metastasis. A total of 25 RCC tissue specimens without metastasis and 13 RCC tissue specimens with bone metastasis were acquired for immunohistochemical analysis of EZH2, MMP2 and TIMP2 protein expression. The expression levels of EZH2, MMP2 and TIMP2 mRNA and protein were analyzed in the ACHN and ACHN-BO5 cell lines using western blot and reverse transcription polymerase chain reaction (PCR) analyses. Methylation-specific PCR was also used to analyze TIMP2 promoter methylation. EZH2 and MMP2 proteins were found to be expressed at higher levels in tissues from patients where RCC had metastasized to the bone as compared with those in RCC patients without metastasis, whereas there was no significant difference in the expression of TIMP2 protein between the two tissues. Furthermore, the expression of EZH2 protein was correlated with MMP2 expression, but there was no significant correlation between the expression of EZH2 and TIMP2 proteins. The in vitro results using cell lines confirmed the ex vivo findings, indicating that the expression levels of EZH2 and MMP2 protein and mRNA were higher in ACHN-BO5 cells than those in ACHN cells. By contrast, TIMP2 protein and mRNA expression levels were lower in ACHN-BO5 cells than those in the parental ACHN cells. The TIMP2 promoter was highly methylated in ACHN-BO5 cells compared with that in ACHN cells. Upregulation of EZH2, MMP2 and TIMP2 expression was correlated with metastasis of RCC to bone tissues ex vivo and in vitro. Further studies are required in order to elucidate the mechanism underlying the altered expression of these genes.

Icariin promotes proliferation and osteogenic differentiation of rat adipose-derived stem cells by activating the RhoA-TAZ signaling pathway

Icariin, the main active flavonoid glucoside isolated from Herba epimedii, has been demonstrated to be a potential alternative therapy to prevent postmenopausal osteoporosis. Icariin has also been shown to regulate the proliferation and osteogenic differentiation of rat bone marrow stromal cells (rBMSCs). However, the detailed molecular mechanism of icariin has remained largely unknown. Besides, no investigation has focused on the relevance of icariin in the regulation of rat adipose-derived stem cells (rASCs) proliferation and osteogenic differentiation. In the present study, we detected that icariin promotes proliferation and osteogenic differentiation of rASCs in a concentration range from 10-8M to 10-6M, with 10-7M to be the optimal concentration. We found that 10-7M icariin significantly increased active RhoA protein expression and ROCK substrate molecule p-MYPT1 expression in rASCs. C3 (the RhoA inhibitor) treatment abrogated the increased proliferation and osteogenic differentiation of rASCs induced by icariin. Interestingly, we also found that C3 abrogated the activation of TAZ induced by icariin. Depletion of TAZ by siRNA transfection significantly blocked icariin promoted proliferation and osteogenic differentiation of rASCs. However, icariin induced active RhoA protein expression was not affected by TAZ specific siRNA transfection, suggesting that RhoA lies upstream of TAZ. Taken together, our data indicate that icariin promotes proliferation and osteogenic differentiation of rASCs by activating the RhoA-TAZ signaling pathway.

Nanog and β-catenin: a new convergence point in EpSC proliferation and differentiation.

Skin tissue homeostasis is maintained by the balanced proliferation and differentiation of certain types of proliferating cells such as epidermal stem cells (EpSCs). The proliferation and differentiation of EpSCs are complex processes which are not well understood. This study aimed to find the internal relationship between the Nanog pathway and the Wnt/β-catenin pathway in the proliferation and differentiation process of EpSCs. In brief, EpSCs were isolated from rat epidermis and cultured. The MTT assay, western blotting, polymerase chain reaction (PCR) and immunocytochemistry were performed during the proliferation and differentiation process of EpSCs. Our results showed that 10−7 M neuropeptide substance P could effectively stimulate proliferation of EpSCs and that a possible link exists between the Nanog pathway and the Wnt/β-catenin pathway.

YAP and ERK mediated mechanical strain-induced cell cycle progression through RhoA and cytoskeletal dynamics in rat growth plate chondrocytes

Yes‐associated protein (YAP) and extracellular signal‐regulated kinase (ERK) have been considered as key regulators in tissue homeostasis, organ development, and tumor formation. However, the roles of YAP and ERK in the mediating strain mechanosensing in the growth plate cartilage have not been determined. In this study, chondrocytes obtained from the growth plate cartilage of 2‐week‐old Sprague–Dawley rats were subjected to the mechanical strain with different magnitudes and durations at a frequency of 0.5 Hz. We found that YAP and ERK activation in response to mechanical strain was time and magnitude dependent. Pretreatment with a RhoA inhibitor (C3 toxin) or a microfilament cytoskeleton disrupting reagent (cytochalasin D) could suppress their activation. In addition, activated YAP and ERK were able to induce cell cycle progression by up‐regulating the expression of cell cycle‐related genes. These results shed new light on the function of YAP and ERK in mechanical strain‐promoted growth plate development. Our results also provided evidence that RhoA and cytoskeletal dynamics are required for this mechanotransduction.