miR-155 Inhibits Mouse Osteoblast Differentiation by Suppressing SMAD5 Expression

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.

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

Microorganisms ◽

10.3390/microorganisms9040673 ◽

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.

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Food for Bone: Evidence for a Role for Delta-Tocotrienol in the Physiological Control of Osteoblast Migration

International Journal of Molecular Sciences ◽

10.3390/ijms21134661 ◽

2020 ◽

Vol 21 (13) ◽

pp. 4661 ◽

Cited By ~ 3

Author(s):

Lavinia Casati ◽

Francesca Pagani ◽

Roberto Maggi ◽

Francesco Ferrucci ◽

Valeria Sibilia

Keyword(s):

Wound Healing ◽

Osteoblast Differentiation ◽

Target Genes ◽

Elaeis Guineensis ◽

Signalling Pathway ◽

Bone Morphogenetic Protein 2 ◽

Luciferase Assay ◽

Boyden Chamber ◽

Differentiation Markers ◽

Osteoblast Migration

Bone remodeling and repair require osteogenic cells to reach the sites that need to be rebuilt, indicating that stimulation of osteoblast migration could be a promising osteoanabolic strategy. We showed that purified δ-tocotrienol (δ-TT, 10 μg/mL), isolated from commercial palm oil (Elaeis guineensis) fraction, stimulates the migration of both MC3T3-E1 osteoblast-like cells and primary human bone marrow mesenchymal stem cells (BMSC) as detected by wound healing assay or Boyden chamber assay respectively. The ability of δ-TT to promote MC3T3-E1 cells migration is dependent on Akt phosphorylation detected by Western blotting and involves Wnt/β-catenin signalling pathway activation. In fact, δ-TT increased β-catenin transcriptional activity, measured using a Nano luciferase assay and pretreatment with procaine (2 µM), an inhibitor of the Wnt/β-catenin signalling pathway, reducing the wound healing activity of δ-TT on MC3T3-E1 cells. Moreover, δ-TT treatment increased the expression of β-catenin specific target genes, such as Osteocalcin and Bone Morphogenetic Protein-2, involved in osteoblast differentiation and migration, and increased alkaline phosphatase and collagen content, osteoblast differentiation markers. The ability of δ-TT to enhance the recruitment of BMSC, and to promote MC3T3-E1 differentiation and migratory behavior, indicates that δ-TT could be considered a promising natural anabolic compound.

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Statin-induced Ras Activation Integrates the Phosphatidylinositol 3-Kinase Signal to Akt and MAPK for Bone Morphogenetic Protein-2 Expression in Osteoblast Differentiation

Journal of Biological Chemistry ◽

10.1074/jbc.m606706200 ◽

2006 ◽

Vol 282 (7) ◽

pp. 4983-4993 ◽

Cited By ~ 100

Author(s):

Nandini Ghosh-Choudhury ◽

Chandi Charan Mandal ◽

Goutam Ghosh Choudhury

Keyword(s):

Bone Morphogenetic Protein ◽

Osteoblast Differentiation ◽

Dominant Negative ◽

Bone Morphogenetic Protein 2 ◽

Regulatory Subunit ◽

Type I ◽

Dependent Manner ◽

Phosphatidylinositol 3 Kinase ◽

Morphogenetic Protein ◽

Phosphatidylinositol 3

Lovastatin promotes osteoblast differentiation by increasing bone morphogenetic protein-2 (BMP-2) expression. We demonstrate that lovastatin stimulates tyrosine phosphorylation of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K), leading to an increase in its kinase activity in osteoblast cells. Inhibition of PI3K ameliorated expression of the osteogenic markers alkaline phosphatase, type I collagen, osteopontin, and BMP-2. Expression of dominant-negative PI3K and PTEN, an inhibitor of PI3K signaling, significantly attenuated lovastatin-induced transcription of BMP-2. Akt kinase was also activated in a PI3K-dependent manner. However, our data suggest involvement of an additional signaling pathway. Lovastatin-induced Erk1/2 activity contributed to BMP-2 transcription. Inhibition of PI3K abrogated Erk1/2 activity in response to lovastatin, indicating the presence of a signal relay between them. We provide, as a mechanism of this cross-talk, the first evidence that lovastatin stimulates rapid activation of Ras, which associates with and activates PI3K in the plasma membrane, which in turn regulates Akt and Erk1/2 to induce BMP-2 expression for osteoblast differentiation.

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Isopsoralen Enhanced Osteogenesis by Targeting AhR/ERα

Molecules ◽

10.3390/molecules23102600 ◽

2018 ◽

Vol 23 (10) ◽

pp. 2600 ◽

Cited By ~ 8

Author(s):

Luna Ge ◽

Yazhou Cui ◽

Kai Cheng ◽

Jinxiang Han

Keyword(s):

Osteoblast Differentiation ◽

Estrogen Receptor Alpha ◽

Biological Effects ◽

Hormone Deficiency ◽

In Vivo Studies ◽

Osteogenic Potential ◽

Type I ◽

Dependent Manner

Isopsoralen (IPRN), one of the main effective ingredients in Psoralea corylifolia Linn, has a variety of biological effects, including antiosteoporotic effects. In vivo studies show that IPRN can increase bone strength and trabecular bone microstructure in a sex hormone deficiency-induced osteoporosis model. However, the mechanism underlying this osteogenic potential has not been investigated in detail. In the present study, we investigated the molecular mechanism of IPRN-induced osteogenesis in MC3T3-E1 cells. Isopsoralen promoted osteoblast differentiation and mineralization, increased calcium nodule levels and alkaline phosphatase (ALP) activity and upregulated osteoblast markers, including ALP, runt-related transcription factor 2 (RUNX2), and collagen type I alpha 1 chain (COL1A1). Furthermore, IPRN limited the nucleocytoplasmic shuttling of aryl hydrocarbon receptor (AhR) by directly binding to AhR. The AhR target gene cytochrome P450 family 1 subfamily A member 1 (CYP1A1) was also inhibited in vitro and in vivo. This effect was inhibited by the AhR agonists indole-3-carbinol (I3C) and 3-methylcholanthrene (3MC). Moreover, IPRN also increased estrogen receptor alpha (ERα) expression in an AhR-dependent manner. Taken together, these results suggest that IPRN acts as an AhR antagonist and promotes osteoblast differentiation via the AhR/ERα axis.

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Betulin Promotes Differentiation of Human Osteoblasts In Vitro and Exerts an Osteoinductive Effect on the hFOB 1.19 Cell Line Through Activation of JNK, ERK1/2, and mTOR Kinases

Molecules ◽

10.3390/molecules24142637 ◽

2019 ◽

Vol 24 (14) ◽

pp. 2637 ◽

Cited By ~ 4

Author(s):

Magdalena Mizerska-Kowalska ◽

Adrianna Sławińska-Brych ◽

Katarzyna Kaławaj ◽

Aleksandra Żurek ◽

Beata Pawińska ◽

...

Keyword(s):

Cell Lines ◽

Osteoblast Differentiation ◽

Mrna Level ◽

Osteogenic Potential ◽

Type I ◽

Differentiation Markers ◽

Alizarin Red ◽

Human Osteoblasts ◽

Osteogenic Supplement

Although betulin (BET), a naturally occurring pentacyclic triterpene, has a variety of biological activities, its osteogenic potential has not been investigated so far. The aim of this study was to assess the effect of BET on differentiation of human osteoblasts (hFOB 1.19 and Saos-2 cells) in vitro in osteogenic (with ascorbic acid as an osteogenic supplement) and osteoinductive (without an additional osteogenic supplement) conditions. Osteoblast differentiation was evaluated based on the mRNA expression (RT-qPCR) of Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), type I collagen-α1 (COL1A1), and osteopontin (OPN). Additionally, ALP activity and production of COL1A1 (western blot analysis) and OPN (ELISA) were evaluated. The level of mineralization (calcium accumulation) was determined with Alizarin red S staining. BET upregulated the mRNA level of RUNX2 and the expression of other osteoblast differentiation markers in both cell lines (except the influence of BET on ALP expression/activity in the Saos-2 cells). Moreover, it increased mineralization in both cell lines in the osteogenic conditions. BET also increased the mRNA level of osteoblast differentiation markers in both cell lines (except for ALP in the Saos-2 cells) in the osteoinductive conditions, which was accompanied with increased matrix mineralization. The osteoinductive activity of BET in the hFOB 1.19 cells was probably mediated via activation of MAPKs (JNK and ERK1/2) and mTOR, as the specific inhibitors of these kinases abolished the BET-induced osteoblast differentiation. Our results suggest that BET has the potential to enhance osteogenesis.

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Taurine Activates BMP-2/Wnt3a-Mediated Osteoblast Differentiation and Mineralization via Akt and MAPK Signaling

Iranian Journal of Public Health ◽

10.18502/ijph.v48i11.3514 ◽

2020 ◽

Author(s):

Minsu PARK ◽

Hyeon Kyeong CHOI ◽

Jeung Hee AN

Keyword(s):

Protein Kinase ◽

Osteoblast Differentiation ◽

Integration Site ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Bone Morphogenetic Protein 2 ◽

Dependent Manner ◽

Ovx Rats

Background: We aimed to elucidate the preventive effects of taurine against osteopenia in ovariectomized (OVX) rats and the mechanisms by which taurine regulates osteoblastogenesis in vitro and in vivo. Methods: The effects of the taurine on human osteoblast MG-63 cell differentiation and osteoblastogenesis effect in OVX rat were examined Konkuk University in 2018 by evaluating osteoblast differentiation, and expression of osteoblast-specific factors by western blotting analysis. Results: Taurine supplementation significantly improved alkaline phosphatase (ALP) activity and mineralization in a concentration-dependent manner. Further, taurine induced the expression of osteogenic growth factors such as bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (RUNX2), small mothers against decapentaplegic 1/5/8 (SMAD1/5/8), wingless-type MMTV integration site family member 3A (Wnt3a), and collagen type 1 (COL-1) via mitogen-activated protein kinase (MAPK) and serine/threonine protein kinase (Akt). Moreover, the RUNX2 activity of the taurine-treated group was enhanced by proteinprotein interactions such as Wnt3a-induced p-AKT/RUNX2 and BMP-mediated SMADs/MAPK/RUNX2 interactions. Conclusion: Our in vitro and in vivo results suggested that taurine can be considered as a potential therapeutic candidate agent for preventing bone loss in postmenopausal osteoporosis.

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

Journal of Bone Metabolism ◽

10.11005/jbm.2021.28.3.223 ◽

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.

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Effects of PIN on Osteoblast Differentiation and Matrix Mineralization through Runt-Related Transcription Factor

International Journal of Molecular Sciences ◽

10.3390/ijms21249579 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9579

Author(s):

Kyung-Ran Park ◽

SooHyun Kim ◽

MyoungLae Cho ◽

Sang Wook Kang ◽

Hyung-Mun Yun

Keyword(s):

Transcription Factor ◽

Cell Migration ◽

Protein Level ◽

Osteoblast Differentiation ◽

Signaling Molecules ◽

Dependent Manner ◽

Alizarin Red ◽

Matrix Mineralization ◽

Related Transcription Factor ◽

Dose Dependent

Styrax Japonica Sieb. et Zucc. has been used as traditional medicine in inflammatory diseases, and isolated compounds have shown pharmacological activities. Pinoresinol glucoside (PIN) belonging to lignins was isolated from the stem bark of S. Japonica. This study aimed to investigate the biological function and mechanisms of PIN on cell migration, osteoblast differentiation, and matrix mineralization. Herein, we investigated the effects of PIN in MC3T3-E1 pre-osteoblasts, which are widely used for studying osteoblast behavior in in vitro cell systems. At concentrations ranging from 0.1 to 100 μM, PIN had no cell toxicity in pre-osteoblasts. Pre-osteoblasts induced osteoblast differentiation, and the treatment of PIN (10 and 30 μM) promoted the cell migration rate in a dose-dependent manner. At concentrations of 10 and 30 μM, PIN elevated early osteoblast differentiation in a dose-dependent manner, as indicated by increases in alkaline phosphatase (ALP) staining and activity. Subsequently, PIN also increased the formation of mineralized nodules in a dose-dependent manner, as indicated by alizarin red S (ARS) staining, demonstrating positive effects of PIN on late osteoblast differentiation. In addition, PIN induced the mRNA level of BMP2, ALP, and osteocalcin (OCN). PIN also upregulated the protein level of BMP2 and increased canonical BMP2 signaling molecules, the phosphorylation of Smad1/5/8, and the protein level of Runt-related transcription factor 2 (RUNX2). Furthermore, PIN activated non-canonical BMP2 signaling molecules, activated MAP kinases, and increased β-catenin signaling. The findings of this study indicate that PIN has biological roles in osteoblast differentiation and matrix mineralization, and suggest that PIN might have anabolic effects in bone diseases such as osteoporosis and periodontitis.

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LncRNA DANCR and miR-320a suppressed osteogenic differentiation in osteoporosis by directly inhibiting the Wnt/β-catenin signaling pathway

Experimental & Molecular Medicine ◽

10.1038/s12276-020-0475-0 ◽

2020 ◽

Vol 52 (8) ◽

pp. 1310-1325

Author(s):

Cheng-Gong Wang ◽

Yi-He Hu ◽

Shi-Long Su ◽

Da Zhong

Keyword(s):

Osteogenic Differentiation ◽

Signaling Pathway ◽

Morphological Changes ◽

Osteoporosis Treatment ◽

Inhibitory Effect ◽

Luciferase Assay ◽

Blue Staining ◽

Alcian Blue Staining ◽

Alizarin Red

Abstract Our study aimed to determine how lncRNA DANCR, miR-320a, and CTNNB1 interact with each other and regulate osteogenic differentiation in osteoporosis. qRT-PCR and western blotting were performed to determine the expression of DANCR, miR-320a, CTNNB1, and the osteoporosis- or Wnt/β-catenin pathway-related markers T-cell factor 1 (TCF-1), runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Interactions between CTNNB1, DANCR, and miR-320a were predicted by bioinformatics approaches and validated using a luciferase assay. Osteoblastic phenotypes were evaluated by ALP staining, ALP activity assay and Alizarin Red staining. The bilateral ovariectomy method was used to establish an in vivo osteoporosis model. Bone morphological changes were examined using hematoxylin and eosin (H&E) and Alcian Blue staining. The expression levels of DANCR and miR-320a in BMSCs derived from osteoporosis patients were upregulated, whereas CTNNB1 expression was downregulated compared with that in healthy controls. Importantly, we demonstrated that miR-320a and DANCR acted independently from each other and both inhibited CTNNB1 expression, whereas the inhibitory effect was additive when miR-320a and DANCR were cooverexpressed. Moreover, we found that DANCR overexpression largely abrogated the effect of the miR-320a inhibitor on CTNNB1 expression and the Wnt/β-catenin signaling pathway in BMSCs during osteogenic differentiation. We further confirmed the results above in BMSCs derived from an osteoporosis animal model. Taken together, our findings revealed that DANCR and miR-320a regulated the Wnt/β-catenin signaling pathway during osteogenic differentiation in osteoporosis through CTNNB1 inhibition. Our results highlight the potential value of DANCR and miR-320a as promising therapeutic targets for osteoporosis treatment.

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Homeobox Protein Msx2 Acts as a Molecular Defense Mechanism for Preventing Ossification in Ligament Fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.24.8.3460-3472.2004 ◽

2004 ◽

Vol 24 (8) ◽

pp. 3460-3472 ◽

Cited By ~ 88

Author(s):

Tatsuya Yoshizawa ◽

Fumio Takizawa ◽

Futabako Iizawa ◽

Osamu Ishibashi ◽

Hiroyuki Kawashima ◽

...

Keyword(s):

Periodontal Ligament ◽

Osteoblast Differentiation ◽

Defense Mechanism ◽

Osteoblastic Differentiation ◽

Bone Morphogenetic Protein 2 ◽

Dependent Manner ◽

Periodontal Ligament Cell ◽

Matrix Mineralization ◽

Histone Deacetylase 1 ◽

Homeobox Protein

ABSTRACT Ligaments and tendons are comprised of tough yet flexible connective tissue. Little is known, however, about the precise characteristics of the cells in ligaments and tendons due to the absence of specific markers and cell lines. We recently reported a periodontal ligament cell line, PDL-L2, with suppressed Runx2/Osf2 transcriptional activity and an inability to form mineralized nodules. The present study demonstrates that the homeobox protein Msx2 is a key factor in suppressing those two functions. Msx2 colocalizes with Runx2/Osf2 and suppresses its activity cooperatively, acting with another corepressor, TLE1, as a complex to recruit histone deacetylase 1 activity. Reverse transcription-PCR and in situ hybridization demonstrated that Msx2 expression is higher in periodontal ligament and tendon cells than in osteoblasts. Stable reduction of Msx2 expression in PDL-L2 cells induces osteoblastic differentiation, thereby causing matrix mineralization. Conversely, stable, forced Msx2 expression in MC3T3-E1 cells prevented osteoblast differentiation and matrix mineralization. Msx2-induced suppression of osteoblast differentiation was repressed by bone morphogenetic protein 2. In addition, Msx2 was downregulated in a symptom- and calcification-dependent manner at the affected region in patients with ossification of the posterior longitudinal ligament. Our findings indicate that Msx2 plays a central role in preventing ligaments and tendons from mineralizing.

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