scholarly journals Probiotic Propionibacterium freudenreichii MJ2 Enhances Osteoblast Differentiation and Mineralization by Increasing the OPG/RANKL Ratio

2021 ◽  
Vol 9 (4) ◽  
pp. 673
Author(s):  
Jiah Yeom ◽  
Seongho Ma ◽  
Young-Hee Lim
Keyword(s):  
Signaling Pathway ◽  
Osteoblast Differentiation ◽  
Surface Proteins ◽  
Bone Morphogenetic Protein 2 ◽  
Ovariectomized Rats ◽  
Osteoblastic Cell ◽  
Enhancement Effect ◽  
Propionibacterium Freudenreichii ◽  
Alizarin Red ◽  
Osteoblastic Cell Line

Osteoblast differentiation is important for the development of bone and the maintenance of bone density. Propionibacterium freudenreichii is a probiotic with an anti-inflammatory property. The aim of this study was to investigate the enhancement effect of P. freudenreichii MJ2 (MJ2) isolated from raw milk on osteoblast differentiation, mineralization, and its signaling pathway. For in vitro and in vivo experiments, human fetal osteoblastic cell line hFOB 1.19 and an ovariectomized rat model were used, respectively. Expression levels of genes and proteins related to osteoblast differentiation and mineralization were measured by real-time polymerase chain reaction (qPCR) and Western blotting, respectively. Alizarin red S staining was performed to measure osteoblast mineralization. Heat-killed MJ2 (hkMJ2)-treated cells showed significantly increased osteoblast differentiation via an increase in the osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL) ratio and significantly increased osteoblast mineralization by stimulating the expression of bone morphogenetic protein 2 and runt-related transcription factor 2. Additionally, oral administration of live or heat-killed MJ2 to ovariectomized rats inhibited osteoporosis-induced bone loss. Specifically, surface proteins isolated from MJ2 promoted osteoblast differentiation and mineralization. In conclusion, MJ2 enhanced osteoblast differentiation and mineralization through the OPG/RANKL signaling pathway and the effective component of MJ2 might be its surface proteins.

scholarly journals Anti-Müllerian Hormone Negatively Regulates Osteoclast Differentiation by Suppressing the Receptor Activator of Nuclear Factor-κB Ligand Pathway

2021 ◽  
Vol 28 (3) ◽  
pp. 223-230
Author(s):  
Jung Ha Kim ◽  
Yong Ryoul Yang ◽  
Ki-Sun Kwon ◽  
Nacksung Kim
Keyword(s):  
Osteoblast Differentiation ◽  
Bone Cells ◽  
Osteoclast Differentiation ◽  
Nuclear Factor Κb ◽  
Bone Morphogenetic Protein 2 ◽  
Tartrate Resistant Acid Phosphatase ◽  
Nuclear Factor ◽  
Bone Homeostasis ◽  
Alizarin Red ◽  
Receptor Activator

Background: Multiple members of the transforming growth factor-β (TGF-β) superfamily have well-established roles in bone homeostasis. Anti-Müllerian hormone (AMH) is a member of TGF-β superfamily of glycoproteins that is responsible for the regression of fetal Müllerian ducts and the transcription inhibition of gonadal steroidogenic enzymes. However, the involvement of AMH in bone remodeling is unknown. Therefore, we investigated whether AMH has an effect on bone cells as other TGF-β superfamily members do.Methods: To identify the roles of AMH in bone cells, we administered AMH during osteoblast and osteoclast differentiation, cultured the cells, and then stained the cultured cells with Alizarin red and tartrate-resistant acid phosphatase, respectively. We analyzed the expression of osteoblast- or osteoclast-related genes using real-time polymerase chain reaction and western blot.Results: AMH does not affect bone morphogenetic protein 2-mediated osteoblast differentiation but inhibits receptor activator of nuclear factor-κB (NF-κB) ligand-induced osteoclast differentiation. The inhibitory effect of AMH on osteoclast differentiation is mediated by IκB-NF-κB signaling.Conclusions: AMH negatively regulates osteoclast differentiation without affecting osteoblast differentiation.

Osteoblastic Cell Differentiation on BMP-2 Pre-Adsorbed Octacalcium Phosphate and Hydroxyapatite

2007 ◽  
Vol 361-363 ◽  
pp. 1025-1028 ◽  
Author(s):  
Takashi Kumagai ◽  
Takahisa Anada ◽  
Yoshitomo Honda ◽  
Masamichi Takami ◽  
Ryutaro Kamijyo ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Room Temperature ◽  
Calcium Phosphates ◽  
Octacalcium Phosphate ◽  
Bone Morphogenetic Protein 2 ◽  
Osteoblastic Cell ◽  
Plastic Plate ◽  
Osteoblastic Cell Line ◽  
Ha Coating ◽  
Morphogenetic Protein

The present study was designed to investigate whether bone morphogenetic protein-2 (BMP-2) adsorbed onto octacalcium phosphate (OCP) and hydroxyapatite (HA) surfaces influences osteoblastic cell differentiation. Osteoblastic cell line, UAMS32, were cultured on plastic plate that was coated of synthetic OCP and HA. BMP-2 was pre-adsorbed on these calcium phosphates at 4°C or room temperature for 24h. The cells more proliferated on the BMP-2 pre-adsorbed OCP and HA than that of the controls (OCP and HA coating without BMP-2). ALP activities were higher on HA than on OCP when BMP-2 were pre-adsorbed at 4°C than at room temperature. The results suggest that pre-adsorption of BMP-2 in different temperature affects osteoblastic cell differentiation, most probably through different adsorption state of BMP-2 on this calcium phosphate.

scholarly journals miR-155 Inhibits Mouse Osteoblast Differentiation by Suppressing SMAD5 Expression

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Yue Gu ◽  
Lianjun Ma ◽  
Lei Song ◽  
Xiaoping Li ◽  
Dong Chen ◽  
...  
Keyword(s):  
Osteoblast Differentiation ◽  
Staining Intensity ◽  
Inhibitory Effect ◽  
Bone Morphogenetic Protein 2 ◽  
Luciferase Assay ◽  
Osteogenic Potential ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Rna Molecules ◽  
Mouse Osteoblast

Osteogenesis from preosteoblasts is important for bone tissue engineering. MicroRNAs are a class of endogenous small RNA molecules that potentially modulate osteogenesis. In this study, we found that miR-155 expression was downregulated in a time-dependent manner in cells of the preosteoblast cell line MC3T3-E1 after osteogenic induction using bone morphogenetic protein 2 (BMP2). Transfection with miR-155 decreased alkaline phosphatase (ALP) activity, ALP expression, and the staining intensity of Alizarin Red in MC3T3-E1 cells treated with BMP2, whereas treatment with miR-155 inhibitor promoted BMP2-induced osteoblast differentiation. The luciferase assay confirmed that miR-155 can bind to the 3′ untranslated region of SMAD5 mRNA. miR-155 transfection significantly decreased the expression of SMAD5 protein and mRNA in MC3T3-E1 cells under control media and the p-SMAD5 protein level during osteogenesis. After transfecting cells with the SMAD5 overexpression plasmids, the inhibitory effect of miR-155 on osteogenesis was significantly attenuated. In conclusion, miR-155 inhibited osteoblast differentiation by downregulating the translation of SMAD5 in mouse preosteoblast cells. Inhibition of miR-155 promoted osteogenic potential and thus it can be used as a potential target in the treatment of bone defects.

Inflammatory cytokines (IL-1α, TNF-α) and LPS modulate the Ca2+signaling pathway in osteoblasts

1998 ◽  
Vol 274 (6) ◽  
pp. C1686-C1698 ◽  
Author(s):  
Vincent K. Tam ◽  
Sandra Schotland ◽  
Jacob Green
Keyword(s):  
Signaling Pathway ◽  
Kinase Inhibitor ◽  
Neonatal Rat ◽  
Osteoblastic Cell ◽  
No Production ◽  
Osteoblastic Cell Line ◽  
Herbimycin A ◽  
Tnf Α ◽  
Intracellular Ca ◽  
Message System

Locally derived growth factors and cytokines in bone play a crucial role in the regulation of bone remodeling, i.e., bone formation and bone resorption processes. We studied the effect of interleukin (IL)-1α, tumor necrosis factor (TNF)-α, and Escherichia coli lipopolysaccharide (LPS) on the hormone-activated Ca2+message system in the osteoblastic cell line UMR-106 and in osteoblastic cultures derived from neonatal rat calvariae. In both cell preparations, IL-1α, TNF-α, and LPS did not alter basal intracellular Ca2+concentration ([Ca2+]i) but attenuated Ca2+transients evoked by parathyroid hormone (PTH) and PGE2in a dose (1–100 ng/ml)- and time (8–24 h)-dependent fashion. The cytokines modulated hormonally induced Ca2+influx (estimated by using Mn2+as a surrogate for Ca2+) as well as Ca2+mobilization from intracellular stores. The latter was linked to suppressed production of hormonally induced inositol 1,4,5-trisphosphate. The effect of cytokines on [Ca2+]iwas abolished by the tyrosine kinase inhibitor herbimycin A (50 ng/ml). The cytokine’s effect was, however, independent of nitric oxide (NO) production, since NO donors (sodium nitroprusside) as well as permeable cGMP analogs augment, rather than attenuate, hormonally induced Ca2+transients in osteoblasts. Given the stimulatory role of cytokines on NO production in osteoblasts, the disparate effects of cytokines and NO on the Ca2+signaling pathway may serve an autocrine/paracrine mechanism for modulating the effect of calciotropic hormones on bone metabolism.

Effects of Vitamin K2 on Bone of Ovariectomized Rats and on a Rat Osteoblastic Cell Line

2002 ◽  
Vol 53 (3) ◽  
pp. 144-148 ◽  
Author(s):  
Ichiro Iwamoto ◽  
Shoichiro Kosha ◽  
Toshinori Fujino ◽  
Yukihiro Nagata
Keyword(s):  
Cell Line ◽  
Vitamin K ◽  
Ovariectomized Rats ◽  
Osteoblastic Cell ◽  
Osteoblastic Cell Line

scholarly journals Melatonin Inhibits Glucose-Induced Apoptosis in Osteoblastic Cell Line Through PERK-eIF2α-ATF4 Pathway

2020 ◽  
Vol 11 ◽  
Author(s):  
Renyi Zhou ◽  
Yue Ma ◽  
Zhengbo Tao ◽  
Shui Qiu ◽  
Zunlei Gong ◽  
...  
Keyword(s):  
Er Stress ◽  
Cell Line ◽  
Signaling Pathway ◽  
High Glucose ◽  
Melatonin Receptors ◽  
Osteoblastic Cell ◽  
Osteoblastic Cell Line ◽  
Translational Activation ◽  
Induced Apoptosis ◽  
Induced Changes

Osteoporosis is a common disease resulting in deteriorated microarchitecture and decreased bone mass. In type 2 diabetes patients, the incidence of osteoporosis is significantly higher accompanied by increased apoptosis of osteoblasts. In this study, using the osteoblastic cell line MC3T3-E1, we show that high glucose reduces cell viability and induces apoptosis. Also, high glucose leads to endoplasmic reticulum (ER) stress (ERS) via an increase in calcium flux and upregulation of the ER chaperone binding immunoglobulin protein (BiP). Moreover, it induces post-translational activation of eukaryotic initiation factor 2 alpha (eIF2α) which functions downstream of PKR-like ER kinase (PERK). This subsequently leads to post-translational activation of the transcription factor 4 (ATF4) and upregulation of C/EBP-homologous protein (CHOP) which is an ER stress-induced regulator of apoptosis, as well as downstream effectors DNAJC3, HYOU1, and CALR. Interestingly, melatonin treatment significantly alleviates the high-glucose induced changes in cell growth, apoptosis, and calcium influx by inhibiting the PERK-eIF2α-ATF4-CHOP signaling pathway. Additionally, the MC3T3-E1 cells engineered to express a phosphodead eIF2α mutant did not show high glucose induced ER stress, confirming that melatonin protects osteoblasts against high-glucose induced changes by decreasing ER-stress induced apoptosis by impacting the PERK-eIF2α-ATF4-CHOP signaling pathway. The protective of melatonin against high glucose-induced ER stress and apoptosis was attenuated when the cells were pre-treated with a melatonin receptor antagonist, indicating that the effect of melatonin was mediated via the melatonin receptors in this context. These findings lay the provide mechanistic insights of melatonin’s protective action on osteoblasts and will be potentially be useful in ongoing pre-clinical and clinical studies to evaluate melatonin as a therapeutic option for diabetic osteoporosis.

In-vitro analysis of rhBMP-2 effects in human osteogenic cells

2013 ◽  
Vol 91 (11) ◽  
pp. 929-934
Author(s):  
Eder M.F. de Oliveira ◽  
Elizabeth F. Martinez ◽  
Jeruza P. Bossonaro ◽  
Renato C. Ribeiro ◽  
Vera C. de Araújo ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
In Vitro Study ◽  
Bone Morphogenetic Protein 2 ◽  
Control Group ◽  
Osteoblastic Cell ◽  
Noncollagenous Proteins ◽  
Western Blot Assay ◽  
Osteoblastic Cell Line

This study evaluated the in vitro expression of bone-related proteins by osteoblasts in the presence of different concentrations of human recombinant bone morphogenetic protein-2 (rhBMP-2). Immortalized human fetal osteoblastic cell line 1.19 (hFOB) were exposed to different concentrations of rhBMP-2 (10, 50, or 100 ng/mL) for 72 h. Cell proliferation and viability (MTT assay), as well as the expression of fibronectin, osteonectin, and osteopontin were assessed by indirect immunofluorescence and Western blot. Neither of the 3 concentrations of rhBMP-2 caused statistically significant alterations in cell proliferation and viability, although the concentration of 100 ng/mL showed lower values for both assays after both 48 and 72 h of exposure. There was no alteration in the expression of noncollagenous proteins, as analyzed by immunofluorescence, when compared with the control group. Furthermore, in the Western blot assay we observed a statistically significant decrease in fibronectin and osteonectin at 100 ng rhBMP-2/mL (p < 0.05) by comparison with the medium alone. The expression of osteopontin decreased slightly in all 3 concentrations of rhBMP-2 tested; however, the change was not statistically significant (p > 0.05). In this in-vitro study, the tested concentrations of rhBMP-2 appeared to decrease the expression of important bone-related molecules in pre-osteoblast cells.

scholarly journals Common Regulation of Growth Arrest and Differentiation of Osteoblasts by Helix-Loop-Helix Factors

2001 ◽  
Vol 21 (21) ◽  
pp. 7416-7428 ◽  
Author(s):  
Noriko Funato ◽  
Kiyoshi Ohtani ◽  
Kimie Ohyama ◽  
Takayuki Kuroda ◽  
Masataka Nakamura
Keyword(s):  
Osteoblast Differentiation ◽  
Growth Arrest ◽  
Serum Starvation ◽  
Osteoblastic Cell ◽  
Specific Gene ◽  
Complete Medium ◽  
Osteoblastic Cell Line ◽  
Helix Loop Helix ◽  
Mg63 Cells ◽  
P21 Promoter

ABSTRACT Cellular differentiation entails the coordination of cell cycle arrest and tissue-specific gene expression. We investigated the involvement of basic helix-loop-helix (bHLH) factors in differentiation of osteoblasts using the human osteoblastic cell line MG63. Serum starvation induced growth arrest at G1 phase, accompanied by expression of cyclin-dependent kinase inhibitor p21WAF1/Cip1. Reporter assays with the p21 gene promoter demonstrated that the combination of E2A (E12 or E47) and coactivator CBP was responsible for p21 induction independent of p53. Twist inhibited E2A-CBP-dependent activation of the exogenous and endogenous p21 promoters. Ids similarly inhibited the exogenously transfected p21 promoter; however less antagonistic effect on the endogenous p21 promoter was observed. Twist was predominantly present in nuclei in MG63 cells growing in complete medium, while it localized mainly in the cytoplasm after serum starvation. The fibroblast growth factor receptor 3 gene (FGFR3), which generates signals leading to differentiation of osteoblasts, was found to be controlled by the same transcriptional regulation as the p21 gene. E2A and Twist influenced alkaline phosphatase expression, a consensus marker of osteoblast differentiation. Expression of E2A and FGFR3 was seen at the location of osteoblast differentiation in the calvaria of mouse embryos, implicating bHLH molecules in physiological osteoblast differentiation. These results demonstrate that a common regulatory system is involved in at least two distinct steps in osteoblastic differentiation. Our results also provide the molecular basis of Saethre-Chotzen syndrome, caused by mutations of the TWISTand FGFR3 genes.

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