MiR-26b-3p regulates osteoblast differentiation via targeting estrogen receptor α

BackgroundUnderstanding of the molecular mechanisms of miRNAs involved in osteoblast differentiation is important for the treatment of bone-related diseases.MethodsMC3T3-E1 cells were induced to osteogenic differentiation by culturing with bone morphogenetic protein 2 (BMP2). After transfected with miR-26b-3p mimics or inhibitors, the osteogenic differentiation of MC3T3-E1 cells was detected by ALP and ARS staining. Cell viability was analyzed by MTT. The expressions of miR-26b-3p and osteogenic related markers and signaling were examined by qPCR and western blot. Direct binding of miR-26b-3p and ER-α were determined by dual luciferase assay.ResultsmiR-26b-3p was significantly down-regulated during osteoblast differentiation. Overexpression of miR-26b-3p inhibited osteoblast differentiation, while inhibition of miR-26b-3p enhanced osteoblast differentiation. Further studies demonstrated miR-26b-3p inhibited the expression of estrogen receptor α (ER-α) by directly targeting to the CDS region of ER-α mRNA. Overexpression of ER-α rescued the suppression effects of miR-26b-3p on osteoblast differentiation, while knockdown of ER-α reversed the upregulation of osteoblast differentiation induced by knockdown of miR-26b-3p.ConclusionOur study demonstrates that miR-26b-3p suppresses osteoblast differentiation of MC3T3-E1 cells via directly targeting ER-α.     

Ethyl acetate and n-butanol fraction of Cissus quadrangularis promotes the mineralization potential of murine pre-osteoblast cell line MC3T3-E1 (sub-clone 4)

In a sequel to investigate osteogenic potential of ethanolic extract of Cissus quadrangularis (CQ), the present study reports the osteoblast differentiation and mineralization potential of ethyl acetate (CQ-EA) and butanol (CQ-B) extracts of CQ on mouse pre-osteoblast cell line MC3T3-E1 (sub-clone 4) with an objective to isolate an antiosteoporotic compound. Growth curve, proliferation, and viability assays showed that both the extracts were nontoxic to the cells even at high concentration (100 µg/ml). The cell proliferation was enhanced at low concentrations (0.1 µg/ml and 1 µg/ml) of both the extracts. They also upregulated the osteoblast differentiation and mineralization processes in MC3T3-E1 cells as reflected by expression profile of osteoblast marker genes such as RUNX2, Osterix, Collagen (COL1A1), Alkaline Phosphatase (ALP), Integrin-related Bone Sialoprotein (IBSP), Osteopontin (OPN), and Osteocalcin (OCN). CQ-EA treatment resulted in early differentiation and mineralization as compared with the CQ-B treatment. These findings suggest that low concentrations of CQ-EA and CQ-B have proliferative and osteogenic properties. CQ-EA, however, is more potent osteogenic than CQ-B.     

p75NTR optimizes the osteogenic potential of human periodontal ligament stem cells by up‐regulating α1 integrin expression

Human periodontal ligament stem cells (hPDLSCs) are a promising source in regenerative medicine. Due to the complexity and heterogeneity of hPDLSCs, it is critical to isolate homogeneous hPDLSCs with high regenerative potential. In this study, p75 neurotrophin receptor (p75NTR) was used to isolate p75NTR⁺ and p75NTR^? hPDLSCs by fluorescence‐activated cell sorting. Differences in osteogenic differentiation among p75NTR⁺, p75NTR^? and unsorted hPDLSCs were observed. Differential gene expression profiles between p75NTR⁺ and p75NTR^? hPDLSCs were analysed by RNA sequencing. α1 Integrin (ITGA1) small interfering RNA and ITGA1‐overexpressing adenovirus were used to transfect p75NTR⁺ and p75NTR^? hPDLSCs. The results showed that p75NTR⁺ hPDLSCs demonstrated superior osteogenic capacity than p75NTR^? and unsorted hPDLSCs. Differentially expressed genes between p75NTR⁺ and p75NTR^? hPDLSCs were highly involved in the extracellular matrix‐receptor interaction signalling pathway, and p75NTR⁺ hPDLSCs expressed higher ITGA1 levels than p75NTR^? hPDLSCs. ITGA1 silencing inhibited the osteogenic differentiation of p75NTR⁺ hPDLSCs, while ITGA1 overexpression enhanced the osteogenic differentiation of p75NTR^? hPDLSCs . These findings indicate that p75NTR optimizes the osteogenic potential of hPDLSCs by up‐regulating ITGA1 expression, suggesting that p75NTR can be used as a novel cell surface marker to identify and purify hPDLSCs to promote their applications in regenerative medicine.     

Ginsenoside Rh2(S) induces the differentiation and mineralization of osteoblastic MC3T3-E1 cells through activation of PKD and p38 MAPK pathways

As part of the search for biologically active anti-osteoporotic agents that enhance differentiation and mineralization of osteoblastic MC3T3-E1 cells, we identified the ginsenoside Rh2(S), which is an active component in ginseng. Rh2(S) stimulates osteoblastic differentiation and mineralization, as manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and Alizarin Red staining, respectively. Rh2(S) activates p38 mitogen-activated protein kinase (MAPK) in time- and concentration-dependent manners, and Rh2(S)-induced differentiation and mineralization of osteoblastic cells were totally inhibited in the presence of the p38 MAPK inhibitor, SB203580. In addition, pretreatment with Go6976, a protein kinase D (PKD) inhibitor, significantly reversed the Rh2(S)-induced p38 MAPK activation, indicating that PKD might be an upstream kinase for p38 MAPK in MC3T3-E1 cells. Taken together, these results suggest that Rh2(S) induces the differentiation and mineralization of MC3T3-E1 cells through activation of PKD/p38 MAPK signaling pathways, and these findings provide a molecular basis for the osteogenic effect of Rh2(S).     

The Effects of Ce on the Proliferation, Osteogenic Differentiation and Mineralization Function of MC3T3-E1 Cells In Vitro

The effects of Ce on the proliferation, osteogenic differentiation and mineralization function of a murine preosteoblast cell line MC3T3-E1 in vitro were investigated at cell and molecular levels. The results showed that Ce promoted the proliferation, osteogenic differentiation and mineralization function of MC3T3-E1 cells at concentrations of 0.0001, 0.001, 0.01, 0.1 and 1???M, but turned to inhibit the proliferation, osteogenic differentiation and mineralization function at concentrations of 10, 100 and 1000???M. Ce displayed the up-regulation of Runx2, BMP2, ALP, BSP, Col I and OCN genes at concentrations of 0.0001 and 0.1???M; these genes were down-regulated in the MC3T3-E1 cells treated with 1000???M Ce. The expression of BMP2, Runx2 and OCN proteins was promoted by Ce at concentrations of 0.0001 and 0.1???M, but these proteins were down-regulated after 1000???M Ce treatment. The results suggest that Ce likely up-regulates or down-regulates the expression of Runx2, which subsequently up- or down-regulates OB marker genes Col I and BMP2 at early stages and ALP and OCN at later stages of differentiation, thus causing to promote or inhibit the proliferation, osteogenic differentiation and mineralization function of MC3T3-E1 cells.     

牙源性间充质干细胞对成骨前体细胞成骨分化的影响

目的:探讨牙源性间充质干细胞对成骨前体细胞成骨分化的影响。方法:将小鼠成骨前体细胞MC3T3-E1分为两组,观察组为牙源性间充质干细胞与MC3T3-E1细胞共培养,对照组为单一MC3T3-E1细胞培养。采用CCK-8法检测细胞增殖水平,采用酶联免疫法检测碱性磷酸酶(Alkaline phosphatase,ALP)活性并进行茜素红染色,采用qRT-PCR、Western blot检测ALP与骨桥素(osteopontin,OPN) m RNA与蛋白表达水平。结果:细胞共培养1 d与3 d后,观察组的细胞增殖指数、ALP活性显著高于对照组(P<0.05)。与对照组相比,观察组的矿化结节显著增加,经茜素红染色呈红褐色。细胞共培养1 d与3 d后,观察组的ALP、OPN m RNA与蛋白相对表达水平显著高于对照组(P<0.05)。结论:牙源性间充质干细胞能促进成骨前体细胞的ALP、OPN表达,提高ALP活性,增加细胞增殖能力,诱发矿化,从而促进成骨分化。     

Osteogenic potential of hexane and dichloromethane fraction of Cissus quadrangularis on murine preosteoblast cell line MC3T3-E1 (subclone 4)

In continuation of the investigation of osteogenic potential of solvent fractions of ethanolic extract of Cissus quadrangularis (CQ), an ancient medicinal plant, most notably known for its bone-healing properties, to isolate and identify antiosteoporotic compounds. In the current study, we report the effect of hexane fraction (CQ-H) and dichloromethane fraction (CQ-D) of CQ on the differentiation and mineralization of mouse preosteoblast cell line MC3T3-E1 (subclone 4). Growth, viability, and proliferation assays revealed that low concentrations (0.1, 1, and 100 ng/ml) of both solvent fractions were nontoxic, whereas higher concentrations were toxic to the cells. Differentiation and mineralization of MC3T3-E1 with nontoxic concentrations of CQ-D and CQ-H revealed that CQ-D delayed the mineralization of MC3T3-E1 cells. However, early and enhanced mineralization was observed in cultures treated with nontoxic concentrations of CQ-H, as indicated by Von Kossa staining and expression profile of osteoblast marker genes such as osterix, Runx2, alkaline phosphatase (ALP), collagen (Col1a1), integrin-related bone sialoprotein (IBSP), osteopontin (OPN), and osteocalcin (OCN). These findings suggest CQ-H as the most efficacious solvent fraction for further investigation to isolate and identify the active compounds in CQ-H.     

甲状旁腺激素对成骨细胞中ClC-3 氯通道表达及成骨分化影响的研究

目的:观察甲状旁腺激素(PTH)对成骨细胞中Cl C-3氯通道表达及成骨分化影响,初步探索Cl C-3介导PTH在细胞成骨分化中的作用。方法:采用10-8M、10-9M、10-10M PTH持续刺激和间断刺激MC3T3-E1细胞72 h后,通过CCK-8试剂盒法检测MC3T3-E1细胞的增殖情况,Real-Time PCR法检测MC3T3-E1细胞中Clcn3及成骨相关基因Alp、Runx2的表达情况,免疫荧光法检测10-9M PTH不同给药方式下对Cl C-3蛋白表达的影响。结果 :经不同浓度PTH连续和间断处理72 h后,结果显示10-9 M PTH间断刺激的MC3T3-E1细胞的增殖能力最强,且其Alp、Runx2 m RNA表达均高于10-8 M组和10-10 M组(P<0.05),而相同浓度间断刺激的MC3T3-E1细胞成骨相关基因的表达均高于持续刺激组,以10-9M间断刺激组差异最显著(P<0.05),而10-8 M和10-10M均无统计学差异(P>0.05),10-9 M PTH刺激的MC3T3-E1细胞中Cl C-3蛋白表达也显著增加(P<0.05)。结论 :成骨细胞的Cl C-3氯通道能够响应PTH的刺激发生变化,并伴随着成骨相关基因Alp、Runx2表达的增强。     

高浓度葡萄糖通过p38MAPK-TXNIP/TRX-ROS通路抑制MC3T3-E1细胞成骨分化

目的：研究高浓度葡萄糖抑制MC3T3-E1细胞成骨分化的机理。方法：建立MC3T3-E1细胞成骨分化诱导体系,观察不同浓度葡萄糖（5.5mM和22mM）对MC3T3-E1细胞成骨分化的影响;用不同浓度的p38 MAPK抑制剂Fr167653（0.1μM、1.0μM和10μM）进行药物干预,观察MC3T3-E1细胞在22mM葡萄糖浓度下成骨分化的变化情况。通过钙含量检测、Real time PCR检测相关分化的变化;用Western Blot方法检测MC3T3-E1细胞分化过程中p38 MAPK磷酸化状态、TXNIP表达水平的变化;使用胰岛素二硫键还原法检测细胞内TRX活性水平;使用活性氧检测试剂盒检测细胞内自由氧生成水平。结果：体外诱导条件下,高浓度（22mM）葡萄糖通过升高p38 MAPK磷酸化水平,上调TXNIP表达水平,同时降低TRX活性,使细胞内自由氧生成增加,抑制MC3T3-E1细胞的成骨分化;Fr167653通过抑制p38 MAPK磷酸化,下调TXNIP表达同时升高TRX活性,抑制细胞内自由氧生成,解除高浓度葡萄糖对细胞成骨分化的抑制作用。结论：高浓度葡萄糖通过p38 MAPK-TXNIP/TRX-ROS信号通路抑制MC3T3-E1细胞成骨分化。     

Chondroitin sulfate-E fine-tunes osteoblast differentiation via ERK1/2, Smad3 and Smad1/5/8 signaling by binding to N-cadherin and cadherin-11

Bone formation in the vertebrate skeleton occurs via the processes of endochondral and membranous ossification. Bone matrices contain chondroitin sulfate (CS) chains that regulate endochondral ossification. However, the function of CS in membranous ossification is unclear. Here, using preosteoblastic MC3T3-E1 cells we demonstrate that chondroitin sulfate-E (CS-E) promotes osteoblast differentiation by binding to both N-cadherin and cadherin-11. Differentiated MC3T3-E1 cells exhibited an increase in the total amount of CS and of E-disaccharide units of CS over time. In addition, CS-E polysaccharide, but not CS-A polysaccharide, bound to N-cadherin and cadherin-11 and enhanced osteoblast differentiation. In contrast, osteoblast differentiation was inhibited in chondroitinase ABC-digested MC3T3-E1 cells. Notably, CS-E polysaccharide and hexasaccharide activated intracellular signaling during osteoblast differentiation in non-contacting MC3T3-E1 cells, decreased ERK1/2 phosphorylation, and activated Smad3 and Smad1/5/8; these reactions were blocked by neutralizing antibodies against N-cadherin or cadherin-11, even though cell-cell adhesion is reported to be required for initiation of MC3T3-E1 cell differentiation. Furthermore, CS-E-unit overexpression in MC3T3-E1 cells increased adhesion of the cells to N-cadherin and cadherin-11, and promoted osteoblast differentiation. Collectively, these results suggest that CS-E is a selective ligand for the potential CS receptors, N-cadherin and cadherin-11, leading to osteoblast differentiation of MC3T3-E1 cells.