scholarly journals miR-452-3p inhibited osteoblast differentiation by targeting Smad4

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12228
Author(s):  
Ming Wu ◽  
Hongyan Wang ◽  
Dece Kong ◽  
Jin Shao ◽  
Chao Song ◽  
...  
Keyword(s):  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Cell Model ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Alizarin Red ◽  
Alp Activity ◽  
Complex Process ◽  
Collagen Type 1

Osteoblast differentiation is a complex process that is essential for normal bone formation. A growing number of studies have shown that microRNAs (miRNAs) are key regulators in a variety of physiological and pathological processes, including osteogenesis. In this study, BMP2 was used to induce MC3T3-E1 cells to construct osteoblast differentiation cell model. Then, we investigated the effect of miR-452-3p on osteoblast differentiation and the related molecular mechanism by RT-PCR analysis, Western blot analysis, ALP activity, and Alizarin Red Staining. We found that miR-452-3p was significantly downregulated in osteoblast differentiation. Overexpression miR-452-3p (miR-452-3p mimic) significantly inhibited the expression of osteoblast marker genes RUNX2, osteopontin (OPN), and collagen type 1 a1 chain (Col1A1), and decreased the number of calcium nodules and ALP activity. In contrast, knockdown miR-452-3p (miR-452-3p inhibitor) produced the opposite effect. In terms of mechanism, we found that Smad4 may be the target of miR-452-3p, and knockdown Smad4 (si-Smad4) partially inhibited the osteoblast differentiation enhanced by miR-452-3p. Our results suggested that miR-452-3p plays an important role in osteoblast differentiation by targeting Smad4. Therefore, miR-452-3p is expected to be used in the treatment of bone formation and regeneration.

scholarly journals Lithium chloride enhances osteoblast differentiation and resists senile osteoporosis

2020 ◽  
Author(s):  
Yizhong Bao ◽  
Xiaoling Lv ◽  
Ying Tang ◽  
Xuanliang Ru ◽  
Jirong Wang ◽  
...  
Keyword(s):  
Bone Formation ◽  
Bone Mass ◽  
Wnt Signaling ◽  
Lithium Chloride ◽  
Osteoblast Differentiation ◽  
Marker Genes ◽  
Mineral Density ◽  
Alizarin Red ◽  
Trabecular Thickness ◽  
Senile Osteoporosis

Abstract Background Lithium chloride (LiCl) is commonly used in the clinic for the treatment of bipolar and other mental disorders. LiCl is an inhibitor of GSK-3β, and has been reported to modulate the balance of adipogenesis and osteogenesis. But, whether LiCl impacts bone formation and homeostasis in senile osteoporosis is still unclear. Methods Analysis of tibia in 2, 5, 7 and 10 months old C57BL/6 male mice were performed by MicroCT (μCT). 7 months old wild-type mice were treated with LiCl orally 0, 100 or 200 mg/kg for 3 months and then tested by μCT. The levels of osteogenesis marker genes and Wnt signaling target genes in bone marrow stromal cells (BMSCs) were detected by reverse transcription quantitative polymerase chain reaction and immunostaining. BMSCs were induced osteoblast differentiation and tested by Alizarin red S staining. Results μCT analyses of C57BL/6 mice showed that bone mineral density (BMD) and trabecular thickness (Tb.Th) increased until the bone mass peaked (5 months) and then began to fall subsequently. LiCl dramatically enhanced bone mass in the senile osteoporotic conditions, represented by increased ratio of bone volume to tissue volume (BV/TV), and decreased in trabeculae separation (Tb.Sp). Moreover, LiCl significantly increased both canonical osteoblastogenesis and Wnt signaling activity without affecting hormones. Conclusion This study uncovered the role of LiCl in canonical Wnt signaling and bone formation and have provided the evidence that LiCl may potentially repress senile osteoporosis.

scholarly journals Aesculetin Promotes Osteoblastogenic Bone Formation Through Enhancing Osteoblast Differentiation and Mineralization

2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 350-350
Author(s):  
WooJin Na ◽  
Young-Hee Kang
Keyword(s):  
Bone Resorption ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Osteoblastic Differentiation ◽  
Alizarin Red S ◽  
Type I ◽  
Mineral Density ◽  
Alizarin Red ◽  
Alp Activity ◽  
Cellular Expression

Abstract Objectives Osteoporosis is a common chronic disease elicited by imbalance between osteoblastic bone formation and osteoclastic bone resorption. Marked increase in bone resorption leads to the aberrant fall in bone mineral density. With increasing age, there is also a significant reduction in bone formation. Aesculetin, a derivative of coumarin, possesses anti-inflammatory and antioxidant effects. The purpose of this study was to identify that aesculetin accelerated bone formation through increased osteoblastic differentiation and mineralization. Methods MC3T3-E1 cells were cultured with 1–10 μM aesculetin in α-MEM supplemented with 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid and 10 μM dexamethasone for up to 21 days. Alkaline phosphatase (ALP) activity and staining, Alizarin red S staining, and Western blotting for induction of target proteins were conducted for the measurement of osteoblastic differentiation and mineralization. Results Aesculetin further enhanced the ALP activity of differentiated MC3T3-E1 cells, showing that aesculetin stimulated the osteoblast differentiation. Alizarin red S staining revealed that calcium deposits highly increased in 1–10 μM aesculetin-treated osteoblasts. In addition, aesculetin further increased cellular expression of the bone-forming markers of bone morphogenetic protein-2, osteopontin and collagen type I in osteoblasts. Conclusions Aesculetin was effective in enhancing osteoblast differentiation and bone mineralization for bone formation, indicating that this compound may be a potential agent for the treatment of osteoporosis. Funding Sources This work was supported by the BK21 FOUR(Fostering Outstanding Universities for Research, 4220200913807) funded by the National Research Foundation of Korea (NRF).

scholarly journals Biochanin A Promotes Osteogenic but Inhibits Adipogenic Differentiation: Evidence with Primary Adipose-Derived Stem Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Shu-Jem Su ◽  
Yao-Tsung Yeh ◽  
Shu-Hui Su ◽  
Kee-Lung Chang ◽  
Huey-Wen Shyu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Formation ◽  
Adipogenic Differentiation ◽  
Biochanin A ◽  
Marker Genes ◽  
Dependent Manner ◽  
Adipose Derived Stem Cells ◽  
Alizarin Red ◽  
Alp Activity ◽  
Cytoplasmic Lipid Droplet

Biochanin A has promising effects on bone formationin vivo, although the underlying mechanism remains unclear yet. This study therefore aimed to investigate whether biochanin A regulates osteogenic and adipogenic differentiation using primary adipose-derived stem cells. The effects of biochanin A (at a physiologically relevant concentration of 0.1–1 μM) were assessedin vitrousing various approaches, including Oil red O staining, Nile red staining, alizarin red S staining, alkaline phosphatase (ALP) activity, flow cytometry, RT-PCR, and western blotting. The results showed that biochanin A significantly suppressed adipocyte differentiation, as demonstrated by the inhibition of cytoplasmic lipid droplet accumulation, along with the inhibition of peroxisome proliferator-activated receptor gamma (PPARγ), lipoprotein lipase (LPL), and leptin and osteopontin (OPN) mRNA expression, in a dose-dependent manner. On the other hand, treatment of cells with 0.3 μM biochanin A increased the mineralization and ALP activity, and stimulated the expression of the osteogenic marker genes ALP and osteocalcin (OCN). Furthermore, biochanin A induced the expression of runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), and Ras homolog gene family, member A (RhoA) proteins. These observations suggest that biochanin A prevents adipogenesis, enhances osteoblast differentiation in mesenchymal stem cells, and has beneficial regulatory effects in bone formation.

scholarly journals Effect of puerarin on osteogenic differentiation of human periodontal ligament stem cells

2019 ◽  
Vol 48 (4) ◽  
pp. 030006051985164
Author(s):  
Jun Li ◽  
Youjian Peng
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Calcium Deposition ◽  
Pcr Analysis ◽  
Cell Surface Markers ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Alp Activity ◽  
Experimental Group

Objective To investigate the effects of the flavonoid, puerarin, on osteogenic differentiation of human periodontal ligament stem cells (PDLSCs). Methods Human PDLSCs were isolated from patients undergoing orthodontic treatment, and the cell surface markers CD146, CD34, CD45, and STRO-1 were identified by immunofluorescence. Cell proliferation was detected by MTT assay; alkaline phosphatase (ALP) activity was measured, and calcium deposition was detected by alizarin red staining. PCR was then used to detect the distributions of COL-I, OPN, Runx2, and OCN, genes related to osteogenic differentiation. Results Staining was positive for cytokines CD146, CD34, CD45, and STRO-1 in the experimental group; staining was also positive for silk protein, but negative for keratin. After 7 days of culture, exposure to puerarin significantly promoted the level of intracellular ALP; increased puerarin concentration led to increased intracellular ALP. Red mineralized nodules appeared upon exposure to puerarin and the number of nodules was concentration-dependent. PCR analysis revealed that COL-I, OPN, Runx2, and OCN expression levels increased as puerarin concentration increased. Conclusions Exposure to puerarin can promote proliferation and ALP activity in human PDLSCs, thus promoting both molecular and osteogenic differentiation; these findings may provide a theoretical basis for the clinical treatment of periodontal disease with puerarin.

scholarly journals ER stress arm XBP1s plays a pivotal role in proteasome inhibition-induced bone formation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Dan Zhang ◽  
Kim De Veirman ◽  
Rong Fan ◽  
Qiang Jian ◽  
Yuchen Zhang ◽  
...  
Keyword(s):  
Bone Formation ◽  
Er Stress ◽  
Osteogenic Differentiation ◽  
Osteoblast Differentiation ◽  
Bone Destruction ◽  
Proteasome Inhibition ◽  
Stress Signaling ◽  
Alizarin Red

Abstract Background Bone destruction is a hallmark of multiple myeloma (MM). It has been reported that proteasome inhibitors (PIs) can reduce bone resorption and increase bone formation in MM patients, but the underlying mechanisms remain unclear. Methods Mesenchymal stem cells (MSCs) were treated with various doses of PIs, and the effects of bortezomib or carfilzomib on endoplasmic reticulum (ER) stress signaling pathways were analyzed by western blotting and real-time PCR. Alizarin red S (ARS) and alkaline phosphatase (ALP) staining were used to determine the osteogenic differentiation in vitro. Specific inhibitors targeting different ER stress signaling and a Tet-on inducible overexpressing system were used to validate the roles of key ER stress components in regulating osteogenic differentiation of MSCs. Chromatin immunoprecipitation (ChIP) assay was used to evaluate transcription factor-promoter interaction. MicroCT was applied to measure the microarchitecture of bone in model mice in vivo. Results We found that both PERK-ATF4 and IRE1α-XBP1s ER stress branches are activated during PI-induced osteogenic differentiation. Inhibition of ATF4 or XBP1s signaling can significantly impair PI-induced osteogenic differentiation. Furthermore, we demonstrated that XBP1s can transcriptionally upregulate ATF4 expression and overexpressing XBP1s can induce the expression of ATF4 and other osteogenic differentiation-related genes and therefore drive osteoblast differentiation. MicroCT analysis further demonstrated that inhibition of XBP1s can strikingly abolish bortezomib-induced bone formation in mouse. Conclusions These results demonstrated that XBP1s is a master regulator of PI-induced osteoblast differentiation. Activation of IRE1α-XBP1s ER stress signaling can promote osteogenesis, thus providing a novel strategy for the treatment of myeloma bone disease.

scholarly journals Strontium ranelate promotes increased peri-implant bone formation in ovariectomized rats.

2020 ◽  
Vol 9 (10) ◽  
pp. e7539109092
Author(s):  
Fernanda Costa Yogui ◽  
Ana Cláudia Ervolino-Silva ◽  
Letícia Pitol-Palin ◽  
Juliana Zorzi Coléte ◽  
Jaqueline Suemi Hassumi ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Bone Formation ◽  
Bone Tissue ◽  
Strontium Ranelate ◽  
Statistical Tests ◽  
Ovariectomized Rats ◽  
Pcr Analysis ◽  
Adult Rats ◽  
Alizarin Red ◽  
Reverse Torque

This study aimed to evaluate the systemic effect of strontium ranelate (SR) on peri-implant bone tissue. Thirty-six adult rats were divided into three experimental groups: sham (SHAM), ovariectomized (OVX) and ovariectomized rats treated with strontium ranelate (OVX-Sr). Strontium ranelate (625mg/kg) was administered by oral gavage on a daily basis. The implants were installed on the tibiae. The euthanasia occurred 42 and 60 days after the implants were installed, and the biomechanical (reverse torque); PCR-RT; histological; immunohistochemical; confocal microscopy and histometric analysis were performed. Quantitative data was subjected to statistical tests with significance level set at p<0.05. Significant increase in implant reverse torque in OVX-Sr was observed when compared to OVX. PCR analysis showed an increase in the genetic expression of the proteins responsible for bone formation in OVX-SR. In the histological analysis, SHAM and OVX-Sr showed a higher degree of maturation of peri-implant bone tissue. Ran-Sr presented higher immunolabeling for ALP and OPN proteins when compared to OVX. In the confocal microscopy, OVX-Sr there was good bone neoformation showed by incorporation of Alizarin red fluorochrome. The histometric analysis, bone implant contact (BIC) and neoformed bone area (NBA) presented statistically difference among all groups, and the Ran-Sr presented the highest BIC. Thus, strontium ranelate improves osseointegration and quality of neoformed bone tissue around implants in estrogen deficient rats.

scholarly journals Collagen Hydrolysate Gly-Pro-Hyp on Osteoblastic Proliferation and Differentiation of MC3T3-E1 Cells

2017 ◽  
Vol 1 (3) ◽  
Author(s):  
Geng Min
Keyword(s):  
Alkaline Phosphatase ◽  
Transcription Factor ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Control Medium ◽  
Collagen Hydrolysate ◽  
Alp Activity ◽  
Proliferation And Differentiation ◽  
Related Transcription Factor ◽  
Set Up

Objectives: Bone formation and bone resorption continuouslyoccur in bone tissue to prevent the accumulation of old bone, thisbeing called bone remodeling. Osteoblasts especially play a crucialrole in bone formation through the differentiation and proliferation.Therefore, in this study, we investigated the effects of collagenhydrolysate Glycine-Prolyl-Hydroxyproline (Gly-Pro-Hyp) onosteoblastic proliferation and differentiation in MC3T3-E1 cells.Methods: Four groups including control, Gly-Pro-Hyp 20 μM,100 μM, 500 μM groups were set up in this study. Cells werecultured with blank control medium or Gly-Pro-Hyp of thedifferent dosages for 24 h. CCK 8 assay was analyzed cellproliferation. Assay of Alkaline phosphatase (ALP)activity wasanalyzed osteoblast differentiation. The expression levels of ALP,Col 1, Runx 2 and Osterix in MC3T3-E1 cells were measured byWestern blot. Results: The results indicated the treatment of Gly-Pro-Hyp promoted the proliferation of MC3T3-E1 cells andimproved ALP activity. In addition, cells treated with Gly-Pro-Hypsignificantly upregulated protein expression of ALP, type 1collagen, runt-related transcription factor 2 and osterix.Conclusion: The results demonstrate that Gly-Pro-Hyp promotedifferentiation inducement and proliferation of MC3T3-E1 cells,therefore may help to elucidate the transcriptional mechanism ofbone formation and possibly lead to the development of bone-forming drugs.

Adenosine Regulates Bone Metabolism Via A1, A2A and A2B Receptors in Bone Marrow Cells From Normal and Patients with Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4977-4977
Author(s):  
Wenjie He ◽  
Jeesun Park ◽  
Amitabha Mazumder ◽  
Bruce Cronstein
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Resorption ◽  
Receptor Agonist ◽  
Osteoblast Differentiation ◽  
Osteoclast Differentiation ◽  
Activity Assay ◽  
Alizarin Red ◽  
Alp Activity ◽  
A2a Receptor

Abstract Abstract 4977 Background: Multiple myeloma is characterized by osteolytic bone lesions, wherein coupled bone remodeling is disrupted with increased osteoclast activation and decreased osteoblast differentiation. We have previously demonstrated that adenosine, acting via A2A receptors, diminishes human and murine osteoclast formation and others have reported that adenosine, acting at A2Breceptors, promotes osteoblast differentiation in murine osteoblast precursors and cell lines. In this study, we examined the effect of adenosine on osteoblast and osteoclast differentiation derived from multiple myeloma (MM) patients. Methods: Human bone marrow was collected from multiple myeloma patients. Bone marrow stromal cells (BMSCs) and bone marrow derived mononuclear (BMMs) cells were isolated and osteoblasts and osteoclastswere cultured, respectively. Adenosine A1 receptor agonist CHA and antagonist Rolofylline, A2A receptor agonist CGS and antagonist ZM, and A2b receptor agonist BAY and antagonist MRS 1754, A3receptor agonist IB-MECA and antagonist MRS 1191; and dipyridamole, a nucleoside transport inhibitor, were added to the culture media. Alkaline phosphatase (ALP) activity assay was used to quantitate the osteoblast differentiation. In vitro osteoblast calcification was determined by alizarin red staining. TRAP+ staining was used to examine the osteoclast differentiation and bone resorption assay was used to study the osteoclast activity. Results: We found that A1R blockade by rolofyllineand A2aR ligation by CGS21680 inhibited differentiation of both normal and MM BMMs into TRAP+ multinucleated cells (IC50= 1nM for A1R, IC50= 10μM for A2AR;p<0. 001, n=3 for both). The inhibition of osteoclast differentiation by Rolofylline was also seen in bone resorption assay (Pit formation assay). The A2A receptor antagonist completely reversed the effects of CGS21680 on osteoclast differentiation. Moreover, enhanced adenosine accumulation in the presence of dipyridamole (0. 5μM) and A2BR activation promoted the differentiation of BMSCs from myeloma patients into osteoblasts shown byArlizarin red staining and ALP activity assay (by 1. 8 ± 0. 41 and 1. 57 ± 0. 26 fold, respectively, p<0. 05, compared with osteogenic media only, n=3 for both). Conclusions: These results indicate that adenosine A2 receptors may be useful targets for the treatment and prevention of MM-induced bone disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Decreased C-Src Expression Enhances Osteoblast Differentiation and Bone Formation

2000 ◽  
Vol 151 (2) ◽  
pp. 311-320 ◽  
Author(s):  
Marilena Marzia ◽  
Natalie A. Sims ◽  
Susanne Voit ◽  
Silvia Migliaccio ◽  
Anna Taranta ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Matrix Protein ◽  
Bone Matrix ◽  
Osteogenic Cells ◽  
Alp Activity ◽  
Pthrp Receptor ◽  
Bone Matrix Protein

c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src−/− relative to Src+/+ mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by ∼60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src−/− osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.

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