u-0126 and Osteoporosis

The objective of this study is to investigate the protective role of hesperetin for the glucocorticoid-induced osteoporosis (GIOP) and related mechanisms. In this study, we investigated the protective effects of hesperetin on dexamethasone (DEX)-induced osteogenic inhibition in bone marrow mesenchymal stem cells (BMSCs). The mineralization, real-time quantitative polymerase chain reaction assays (RT-qPCR), immunofluorescence and western blot were used to assess the protective effects of hesperetin in DEX-treated BMSCs during osteogenic differentiation. Our results showed that hesperetin promoted alkaline phosphatase (ALP) activity and the mineralization in DEX-treated BMSCs during osteogenic differentiation. The expression of osteogenic mRNA and proteins further confirmed the protective effect of hesperetin in DEX-treated BMSCs. Furthermore, hesperetin activated ERK signal pathway in DEX-treated BMSCs. ERK inhibitor U0126 could abolish the protective effect of hesperein in DEX-treated BMSCs. In conclusion, our study demonstrated that hesperetin alleviated glucocorticoid-induced inhibition of osteogenic differentiation through ERK signal pathway in BMSCs. It may be a potential therapeutic agent for protecting against glucocorticoid-induced osteoporosis.

Topics: Butadienes; Cell Differentiation; Dexamethasone; Extracellular Signal-Regulated MAP Kinases; Glucocorticoids; Hesperidin; Humans; MAP Kinase Signaling System; Mesenchymal Stem Cells; Nitriles; Osteogenesis; Osteoporosis; Primary Cell Culture

Myricetin has been reported to promote osteogenic differentiation and inhibit osteoclastogenesis. The aim of this study was to investigate the potential effects of myricetin on glucocorticoid-induced osteoporosis (GIOP) both in vivo and in vitro. Male Sprague-Dawley (SD) rats were given dexamethasone (DEX, 0.1 mg/kg, s.c.) once daily and myricetin (1 mg/kg or 2.5 mg/kg, i.p.) once every other day for a total of five weeks. Body weight was recorded once a week. Bone mineral density (BMD), the activities or levels of bone turnover markers, and histological changes were assessed. MC3T3-E1 cells were incubated with DEX (1 μM) and myricetin (20 μM). Osteoblast proliferation, differentiation and mineralization were evaluated. U0126 was added to evaluate the involvement of the ERK signaling pathway. The results showed that myricetin increased body weight gain and inhibited DEX-induced reduction in BMD, enhanced alkaline phosphatase (ALP) activity, and upregulated osteocalcin (OCN), bone morphogenetic protein 2 (BMP2) and runt-related transcription factor 2 (Runx2) levels, whereas reduced tartrate-resistant acid phosphatase (TRAP) activity and C-terminal telopeptide of type I collagen (CTx) level. In addition, myricetin ameliorated histological changes in the femurs. In our in vitro studies, myricetin promoted osteoblast differentiation and mineralization in DEX-treated MC3T3-E1 cells, accompanied by increases in BMP2, Runx2, ALP, OCN, collagen type I alpha 1 (COL1A1) and osteopontin (OPN) levels. The promotion effects of myricetin on osteogenic differentiation and matrix mineralization were reversed by U0126. These results suggest that myricetin may alleviate DEX-induced osteoporosis by promoting osteogenic differentiation and matrix mineralization via the ERK signaling pathway.

Topics: 3T3 Cells; Alkaline Phosphatase; Animals; Body Weight; Bone Morphogenetic Protein 2; Butadienes; Cell Differentiation; Cell Proliferation; Core Binding Factor Alpha 1 Subunit; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Glucocorticoids; Male; MAP Kinase Signaling System; Mice; Nitriles; Osteoblasts; Osteocalcin; Osteogenesis; Osteoporosis; Rats; Rats, Sprague-Dawley; Signal Transduction

Osteoporosis (OP) is a serious health problem that contributes to osteoporotic structural damage and bone fragility. MicroRNAs (miRNAs) can exert important functions over bone endocrinology. Therefore, it is of substantial significance to clarify the expression and function of miRNAs in bone endocrine physiology and pathology to improve the potential therapeutic value for metabolism-related bone diseases. We explored the effect of microRNA-182-5p (miR-182-5p) on osteoblast proliferation and differentiation in OP rats after alendronate (ALN) treatment by targeting adenylyl cyclase isoform 6 (ADCY6) through the Rap1/mitogen-activated protein kinase (MAPK) signaling pathway. Rat models of OP were established to observe the effect of ALN on OP, and the expression of miR-182-5p, ADCY6 and the Rap1/MAPK signaling pathway-related genes was determined. To determine the roles of miR-182-5p and ADCY6 in OP after ALN treatment, the relationship between miR-182 and ADCY6 was initially verified. Osteoblasts were subsequently extracted and transfected with a miR-182-5p inhibitor, miR-182-5p mimic, si-ADCY6 and the MAPK signaling pathway inhibitor U0126. Cell proliferation, apoptosis and differentiation were also determined. ALN treatment was able to ease the symptoms of OP. miR-182-5p negatively targeted ADCY6 to inhibit the Rap1/MAPK signaling pathway. Cells transfected with miR-182 inhibitor decreased the expression of ALP, BGP and COL I, which indicated that the down-regulation of miR-182-5p promoted cell differentiation and cell proliferation and inhibited cell apoptosis. In conclusion, the present study shows that down-regulated miR-182-5p promotes the proliferation and differentiation of osteoblasts in OP rats through Rap1/MAPK signaling pathway activation by up-regulating ADCY6, which may represent a novel target for OP treatment.

Topics: Adenylyl Cyclases; Alendronate; Animals; Butadienes; Cell Differentiation; Cell Proliferation; Gene Expression Regulation; Humans; MAP Kinase Kinase 1; MicroRNAs; Nitriles; Osteoblasts; Osteoporosis; Rats; Shelterin Complex; Signal Transduction; Telomere-Binding Proteins

Angiogenesis plays an important role in bone development and remodeling and is mediated by a plethora of potential angiogenic factors. However, data regarding specific angiogenic factors that are secreted within the bone microenvironment to regulate osteoporosis is lacking. Here, we report that Nephronectin (NPNT), a member of the epidermal growth factor (EGF) repeat superfamily proteins and a homologue of EGFL6, is expressed in osteoblasts. Intriguingly, the gene expression of NPNT is reduced in the bone of C57BL/6J ovariectomised mice and in osteoporosis patients. In addition, the protein levels of NPNT and CD31 are also found to be reduced in the tibias of OVX mice. Exogenous addition of mouse recombinant NPNT on endothelial cells stimulates migration and tube-like structure formation in vitro. Furthermore, NPNT promotes angiogenesis in an ex vivo fetal mouse metatarsal angiogenesis assay. We show that NPNT stimulates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated kinase (MAPK) in endothelial cells. Inhibition of ERK1/2 impaired NPNT-induced endothelial cell migration, tube-like structure formation and angiogenesis. Taken together, these results demonstrate that NPNT is a paracrine angiogenic factor and may play a role in pathological osteoporosis. This may lead to new targets for treatment of bone diseases and injuries.

Topics: Animals; Butadienes; Cell Differentiation; Cells, Cultured; Down-Regulation; Extracellular Matrix Proteins; Female; Humans; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Neovascularization, Physiologic; Nitriles; Osteoblasts; Osteoclasts; Osteoporosis; Ovariectomy; Platelet Endothelial Cell Adhesion Molecule-1; Transcriptome

Protein tyrosine phosphatases (PTPs) are pivotal regulators of key cellular functions, including cell growth, differentiation, and adhesion. Previously, we reported that leukocyte common antigen-related (LAR) tyrosine phosphatase promotes osteoblast differentiation in MC3T3-E1 preosteoblast cells. In the present study, the mechanism of the regulatory action of LAR on osteoblast differentiation was investigated. The mineralization of extracellular matrix and calcium accumulation in MC3T3-E1 cells were markedly enhanced by LAR overexpression, and these effects were further increased by treatment with a MEK inhibitor. In addition, LAR overexpression dramatically reduced extracellular signal-regulated kinase (Erk) activation during osteoblast differentiation. In contrast, a marginal effect of the inactive LAR mutant on Erk activation was detected. Expression of osteoblast-related genes such as ALP, BSP, DLX5, OCN, and RUNX2, was increased by LAR overexpression during osteoblast differentiation. On the basis of these results, we propose that LAR functions as a positive regulator of osteoblast differentiation by modulating ERK activation. Therefore, LAR phosphatase could be used as a novel regulatory target protein in many bone-associated diseases, including osteoporosis.

Topics: Animals; Butadienes; Calcification, Physiologic; Calcium; Cell Differentiation; Cell Line; Extracellular Matrix; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Developmental; HLA Antigens; Humans; Mice; Mitogen-Activated Protein Kinases; Nitriles; Osteoblasts; Osteogenesis; Osteoporosis; Phosphorylation; Protein Tyrosine Phosphatases; Receptor-Like Protein Tyrosine Phosphatases, Class 4; Signal Transduction; Transfection