Mouse Osteoblastic MC3T3-E1 Cells Research Articles

Understanding the functional role of genistein in the bone differentiation in mouse osteoblastic cell line MC3T3-E1 by RNA-seq analysis

Genistein, a phyto-estrogen, can potentially replace endogenous estrogens in postmenopausal women, but the underlying molecular mechanisms remain incompletely understood. To obtain insight into the effect of genistein on bone differentiation, RNA sequencing (RNA-seq) analysis was used to detect differentially expressed genes (DEGs) in genistein-treated vs. untreated MC3T3-E1 mouse osteoblastic cells. Osteoblastic cell differentiation was monitored by measuring osteoblast differentiation factors (ALP production, bone mineralization, and expression of osteoblast differentiation markers). From RNA-seq analysis, a total of 132 DEGs (including 52 up-regulated and 80 down-regulated genes) were identified in genistein-treated cells (FDR q-value < 0.05 and fold change > 1.5). KEGG pathway and Gene Ontology (GO) enrichment analyses were performed to estimate the biological functions of DEGs and demonstrated that these DEGs were highly enriched in functions related to chemotactic cytokines. The functional relevance of DEGs to genistein-induced osteoblastic cell differentiation was further evaluated by siRNA-mediated knockdown in MC3T3-E1 cells. These siRNA knockdown experiments (of the DEGs validated by real-time qPCR) demonstrated that two up-regulated genes (Ereg and Efcab2) enhance osteoblastic cell differentiation, while three down-regulated genes (Hrc, Gli, and Ifitm5) suppress the differentiation. These results imply their major functional roles in bone differentiation regulated by genistein.

Selective Androgen Receptor Modulator, YK11, Up-Regulates Osteoblastic Proliferation and Differentiation in MC3T3-E1 Cells.

Androgens are key regulators that play a critical role in the male reproductive system and have anabolic effects on bone mineral density and skeletal muscle mass. We have previously reported that YK11 is a novel selective androgen receptor modulator (SARM) and induces myogenic differentiation and selective gene regulation. In this study, we show that treatment of YK11 and dihydrotestosterone (DHT) accelerated cell proliferation and mineralization in MC3T3-E1 mouse osteoblast cells. Further, YK11-treated cells increased osteoblast specific differentiation markers, such as osteoprotegerin and osteocalcin, compared to untreated cells. These observations were attenuated by androgen receptor (AR) antagonist treatment. To clarify the effect of YK11, we investigated rapid non-genomic signaling by AR. The phosphorylated Akt protein level was increased by YK11 and DHT treatment, suggesting that YK11 activates Akt-signaling via non-genomic signaling of AR. Because it is known Akt-signaling is a key regulator of androgen-mediated osteoblast differentiation, YK11 has osteogenic activity as well as androgen.

Magnesium phosphate ceramics incorporating a novel indene compound promote osteoblast differentiation in vitro and bone regeneration in vivo

Incorporating bioactive molecules into synthetic ceramic scaffolds is challenging. In this study, to enhance bone regeneration, a magnesium phosphate (MgP) ceramic scaffold was incorporated with a novel indene compound, KR-34893. KR-34893 induced the deposition of minerals and expression of osteoblast marker genes in primary human bone marrow mesenchymal stem cells (BMSCs) and a mouse osteoblastic MC3T3-E1 cell line. Analysis of the mode of action showed that KR-34893 induced the phosphorylation of MAPK/extracellular signal-regulated kinase and extracellular signal-regulated kinase, and subsequently the expression of bone morphogenetic protein 7, accompanied by SMAD1/5/8 phosphorylation. Accordingly, KR-34893 was incorporated into an MgP scaffold prepared by 3D printing at room temperature, followed by cement reaction. KR-34893-incorporated MgP (KR-MgP) induced the expression of osteoblast differentiation marker genes in vitro. In a rat calvaria defect model, KR-MgP scaffolds enhanced bone regeneration and increased bone volume compared with MgP scaffolds, as assessed by micro-computed tomography and histological analyses. In conclusion, we developed a method for producing osteoinductive MgP scaffolds incorporating a bioactive organic compound, without high temperature sintering. The KR-MgP scaffolds enhanced osteoblast activation in vitro and bone regeneration in vivo.

Inhibition of microRNA-214-5p promotes cell survival and extracellular matrix formation by targeting collagen type IV alpha 1 in osteoblastic MC3T3-E1 cells.

ObjectivesThis study aimed to investigate the functional effects of microRNA (miR)-214-5p on osteoblastic cells, which might provide a potential role of miR-214-5p in bone fracture healing.MethodsBlood samples were obtained from patients with hand fracture or intra-articular calcaneal fracture and from healthy controls (HCs). Expression of miR-214-5p was monitored by qRT-PCR at day 7, 14 and 21 post-surgery. Mouse osteoblastic MC3T3-E1 cells were transfected with antisense oligonucleotides (ASO)-miR-214-5p, collagen type IV alpha 1 (COL4A1) vector or their controls; thereafter, cell viability, apoptotic rate, and the expression of collagen type I alpha 1 (COL1A1), type II collagen (COL-II), and type X collagen (COL-X) were determined. Luciferase reporter assay, qRT-PCR, and Western blot were performed to ascertain whether COL4A1 was a target of miR-214-5p.ResultsPlasma miR-214-5p was highly expressed in patients with bone fracture compared with HCs after fracture (p < 0.05 or p < 0.01). Inhibition of miR-214-5p increased the viability of MC3T3-E1 cells and the expressions of COL1A1 and COL-X, but decreased the apoptotic rate and COL-II expression (p < 0.05 or p < 0.01). COL4A1 was a target of miR-214-5p, and was negatively regulated by miR-214-5p (p < 0.05 or p < 0.01). Overexpression of COL4A1 showed a similar impact on cell viability, apoptotic rate, and COL1A1, COL-II, and COL-X expressions inhibiting miR-214-5p (p < 0.01).ConclusionInhibition of miR-214-5p promotes cell survival and extracellular matrix (ECM) formation of osteoblastic MC3T3-E1 cells by targeting COL4A1.Cite this article: Q. S. Li, F. Y. Meng, Y. H. Zhao, C. L. Jin, J. Tian, X. J. Yi. Inhibition of microRNA-214-5p promotes cell survival and extracellular matrix formation by targeting collagen type IV alpha 1 in osteoblastic MC3T3-E1 cells. Bone Joint Res 2017;6:464–471. DOI: 10.1302/2046-3758.68.BJR-2016-0208.R2

Composite electrospun nanofibers of reduced graphene oxide grafted with poly(3-dodecylthiophene) and poly(3-thiophene ethanol) and blended with polycaprolactone

In this paper, an effective method was employed for preparation of nanofibers using conducting polymer-functionalized reduced graphene oxide (rGO). First, graphene oxide (GO) was obtained from graphite by Hommer method. GO was reduced to rGO by NaBH4 and covalently functionalized with a 3-thiophene acetic acid (TAA) by an esterification reaction to reach 3-thiophene acetic acid-functionalized reduced graphene oxide macromonomer (rGO-f-TAAM). Afterward, rGO-f-TAAM was copolymerized with 3-dodecylthiophene (3DDT) and 3-thiophene ethanol (3TEt) to yield rGO-f-TAA-co-PDDT (rGO-g-PDDT) and rGO-f-TAA-co-P3TEt (rGO-g-PTEt), which were confirmed by Fourier transform infrared spectra. The grafted materials depicted better electrochemical properties and superior solubilities in organic solvents compared to GO and rGO. The soluble rGO-g-PDDT and rGO-g-PTEt composites blended with polycaprolactone were fabricated by electrospinning, and then cytotoxicity, hydrophilicity, biodegradability and mechanical properties were investigated. The grafted rGO composites exhibited a good electroactivity behavior, mainly because of the enhanced electrochemical performance. The electrospun nanofibers underwent degradation about 7 wt% after 40 days, and the fabricated scaffolds were not able to induce cytotoxicity in mouse osteoblast MC3T3-E1 cells. The soluble conducting composites developed in this study are utilizable in the fabrication of nanofibers with tissue engineering application.

Role of autophagy in tumor necrosis factor-α-induced apoptosis of osteoblast cells

The aim of this study is to investigate the role of tumor necrosis factor-α (TNF-α) in apoptosis and autophagy of mouse osteoblast MC3T3-E1 cells, as well as the crosstalk between...

Osthole Enhances Osteogenesis in Osteoblasts by Elevating Transcription Factor Osterix via cAMP/CREB Signaling In Vitro and In Vivo.

Anabolic anti-osteoporotic agents are desirable for treatment and prevention of osteoporosis and fragility fractures. Osthole is a coumarin derivative extracted from the medicinal herbs Cnidium monnieri (L.) Cusson and Angelica pubescens Maxim.f. Osthole has been reported with osteogenic and anti-osteoporotic properties, whereas the underlying mechanism of its benefit still remains unclear. The objective of the present study was to investigate the osteopromotive action of osthole on mouse osteoblastic MC3T3-E1 cells and on mouse femoral fracture repair, and to explore the interaction between osthole-induced osteopromotive effect and cyclic adenosine monophosphate (cAMP) elevating effect. Osthole treatment promoted osteogenesis in osteoblasts by enhancing alkaline phosphatase (ALP) activity and mineralization. Oral gavage of osthole enhanced fracture repair and increased bone strength. Mechanistic study showed osthole triggered the cAMP/CREB pathway through the elevation of the intracellular cAMP level and activation of the phosphorylation of the cAMP response element-binding protein (CREB). Blockage of cAMP/CREB downstream signals with protein kinase A (PKA) inhibitor KT5720 partially suppressed osthole-mediated osteogenesis by inhibiting the elevation of transcription factor, osterix. In conclusion, osthole shows osteopromotive effect on osteoblasts in vitro and in vivo. Osthole-mediated osteogenesis is related to activation of the cAMP/CREB signaling pathway and downstream osterix expression.

Icaritin promotes maturation and mineralization of mouse osteoblast MC3T3-E1 cells through CXCR4/SDF-1 signal pathway

<b>Objective:</b> To investigate the effect of icaritin on maturation and mineralization of mouse osteoblast MC3T3-E1 cells and its mechanism. <b>Methods:</b> The cultured MC3T3-E1 cells were divided into blank control group, CXC chemokine receptor type 4 (CXCR4) inhibitor (AMD3100) group, icaritin group, and icaritin plus AMD3100 group. The expression of CXCR4, stromal cell-derived factor 1 (SDF-1) and osteogenesis-related genes and proteins were detected by real-time RT-PCR and Western blotting after drug treatment for 24 h. The alkaline phosphatase (ALP) activity was determined with ALP kit on d3 and d6; calcium nodules were detected by alizarin red staining after drug treatment for 14 d. <b>Results:</b> Real time RT-PCR showed that compared with the blank control group, relative expressions of <i>CXCR4, SDF-1</i> and osteogenesis-related genes in icaritin group were significantly increased (<i>P</i><0.05 or <i>P</i><0.01); After AMD3100 treatment, the relative expression of <i>CXCR4</i> gene was decreased (<i>P</i><0.05). Western blot showed that compared with the blank control group, relative expressions of CXCR4, SDF-1 and osteogenesis-related proteins in the icaritin group were significantly increased (all <i>P</i><0.01), but were decreased after AMD3100 was added (all <i>P</i><0.01). The ALP activity of icaritin group was significantly higher than that of blank control group (all <i>P</i><0.01) on d3 and d6 after drug treatment, while the activity of ALP was significantly decreased after AMD3100 treatment (all <i>P</i><0.01). At d14 after drug treatment, compared with the blank control group, the area of alizarin red staining was increased in the icaritin group, while it was significantly reduced after the addition of AMD3100. <b>Conclusion:</b> Icaritin may promote maturation and mineralization of mouse osteoblast MC3T3-E1 cells through CXCR4/SDF-1 signaling pathway.

Multilayered co-electrospun scaffold containing silver sulfadiazine as a prophylactic against osteomyelitis: Characterization and biological in vitro evaluations

Bone related-bacterial diseases including wound infections and osteomyelitis (OM) still remain a serious problem. In this study, a hybrid co-electrospun membrane consisting of gelatin (GE) and Poly(d,l-lactide-co-glycolide) (PLGA) fibrous sheets containing different concentrations (0, 0.1, 0.5, and 1wt%) of silver sulfadiazine (AgSD) was designed to provide for improved antimicrobial effect and biocompatibility. Well-defined products were characterized by physicochemical analyses. For biological in vitro assessments, mouse osteoblastic MC3T3-E1 cells were cultured on the scaffolds. This test was done in order to assay for cytotoxicity by measuring cell proliferation. Antibacterial activity against gram-negative Pseudomonas aeruginosa (P. aeruginosa), gram-positive Staphylococcus aureus (S. aureus), and Methicillin-resistant Staphylococcus aureus (MRSA) was also tested. These biological tests showed that GE/PLGA-AgSD scaffolds had good cell viability, as well as effective antimicrobial activity. These remarkable results suggest that GE/PLGA-AgSD scaffolds possess great potential for the treatment of OM and can find many uses in the field of bone tissue engineering.

Hen egg yolk phosvitin stimulates osteoblast differentiation in the absence of ascorbic acid.

Egg yolk phosvitin, one of the most highly phosphorylated extracellular matrix proteins known in nature, has a strong calcium binding and reducing capacity. Here, we investigated the effects of phosvitin on osteoblast differentiation and osteogenic gene expression in cultured mouse osteoblastic MC3T3-E1 cells by using alkaline phosphatase activity analysis, alizarin red S staining and real-time PCR assay. Alkaline phosphatase activity and alizarin red S staining analyses demonstrated no significant difference between differentiating MC3T3-E1 cells cultured in the presence of phosvitin and those cultured in the presence of ascorbic acid after 21 days of differentiation. Our real-time PCR assay also indicated the two groups were similar in the expression of the osteogenic gene markers, collagen type I, osteocalcin, runt-related transcription factor 2, and bone morphogenetic protein-2. Our findings indicate that phosvitin plays a similar role to that of ascorbic acid in osteoblast differentiation and mineralisation. © 2017 Society of Chemical Industry.

Effects of Brucine on the OPG/RANKL/RANK Signaling Pathway in MDA-MB-231 and MC3T3-E1 Cell Coculture System.

The present study examined the effects of brucine on the OPG/RANKL/RANK signaling pathway for exploring the mechanism of brucine suppression of bone metastasis in breast cancer. MDA-MB-231 breast cancer cells and mouse osteoblast MC3T3-E1 cells were cocultured to mimic the breast cancer bone metastasis microenvironment in vitro. qRT-PCR and Western blotting were used to detect the expressions of OPG and RANKL at the mRNA and protein levels, respectively, in brucine-treated cultures and they were compared to those in untreated cultures. We aimed to understand the effect of brucine on the entire OPG/RANKL/RANK signaling pathway after analyzing these effects. Results showed that brucine treatment significantly increased both the OPG mRNA/RANKL mRNA expression ratio and the OPG protein/RANKL protein ratio in cocultures compared to those in untreated cocultures (P < 0.01). Brucine, therefore, plays a regulatory role in the OPG/RANKL/RANK signaling pathway, suggesting that it can indirectly control osteoclasts by regulating the expression and secretion of OPG and RANKL in osteoblast cells, thereby inhibiting the differentiation and bone resorption function of osteoclasts.

In vitro evaluation of biocompatibility of Ti–Mo–Sn–Zr superelastic alloy

Shape memory alloys (SMAs) including superelastic alloys have unique properties such as shape memory and superelasticity, thus they are recognized as very useful biomaterials. These properties are very advantageous for medical use, and actually the SMA wires have been widely used in medical field. However, biocompatibility of nickel-titanium (Ni-Ti) alloy, which is the only practical SMA at present, has been questioned because of its high nickel content. The aim of this study was to evaluate the biocompatibility of a newly developed Ni-free Ti-based SMA for medical use. The newly developed SMA made of Ti-Mo-Sn-Zr system was processed into a disk of 15.1 mm in diameter. Pure titanium of the same shape was prepared as control. All the disk surfaces were polished using emery papers, #120, #400, and #600. Scanning electron microscopy and a 3D optics profiler were used to evaluate the surface of the materials. In vitro evaluations included colony examination for evaluation of the cell cytotoxicity, DNA quantification for the cell proliferation, Alamar blue assay for metabolic activity, FDA staining for the live cell imaging, and cell cycle analysis, using Chinese hamster fibroblastic V-79 cells and mouse osteoblastic MC3T3-E1 cells. In colony examination and DNA quantification, there was no significant difference between the Ti-Mo-Sn-Zr and the pure titanium. In FDA staining, cultured cells on the Ti-Mo-Sn-Zr alloy showed the same biocompatibility as those on the pure titanium. The present results suggest that the newly developed Ti-Mo-Sn-Zr alloy showed the high biocompatibility comparable to pure titanium and can be used as efficient biomaterial for medical use.

Electrospun homogeneous silk fibroin/poly (ɛ-caprolactone) nanofibrous scaffolds by addition of acetic acid for tissue engineering.

In this study, we investigated the phase separation phenomenon of silk fibroin/poly (ɛ-caprolactone) electrospinning solution to improve the performance of silk fibroin/poly (ɛ-caprolactone) electrospun nanofibers. It showed that phase separation does occur in just a few hours in the silk fibroin/poly (ɛ-caprolactone)/formic acid mixture solution. Acetic acid, small molecule nonsolvent for silk fibroin, was first introduced to silk fibroin/poly (ɛ-caprolactone)/formic acid solution, a homogeneous solution without separation for over several days was achieved after mixing for 5 h. The morphology and composition of the silk fibroin/poly (ɛ-caprolactone) and acetic acid-modified silk fibroin/poly (ɛ-caprolactone) fibrous scaffolds were examined by scanning electron microscopy, Fourier transform infrared spectroscopy and thermal gravimetric analyzer. Attachment and proliferation of mouse osteoblast MC3T3-E1 cells were tested by scanning electron microscopy and cytotoxity assay. The results indicated that the phase separation of silk fibroin/poly (ɛ-caprolactone) solution might led to inhomogeneous morphology and composition of the composite scaffolds, and the inhomogeneity of the silk fibroin/poly (ɛ-caprolactone) scaffolds with formic acid as solvent had a remarkable difference on cell adhesion and proliferation. In contrast, there was no significant difference among the silk fibroin/poly (ɛ-caprolactone) scaffolds with formic acid/acetic acid as solvent because of their good consistency in fiber morphology and composition. These obtained silk fibroin/poly (ɛ-caprolactone) nanofibers had small average diameter of 190 ± 40 nm. The obtained results proved that this study provided a facile and effective approach to achieve compositionally homogeneous silk fibroin/poly (ɛ-caprolactone) scaffolds with formic acid as solvent for effective applications.

Preparation and in vitro evaluation of a biomimetic nanoscale calcium phosphate coating on a polyethylene terephthalate artificial ligament.

In the present study, a polyethylene terephthalate (PET) artificial ligament was coated with an organic layer-by-layer (LBL) self-assembled template of chitosan and hyaluronic acid, and then incubated in a calcium phosphate (CaP) solution to prepare a biomimetic CaP coating. The surface characterization of the ligament was examined using scanning electron microscopy, atomic force microscopy and energy-dispersive X-ray spectroscopy. The effects of CaP coatings on the osteogenic activity of MC3T3 E1 mouse osteoblastic cells were investigated by evaluating their attachment, proliferation and the relative expression levels of alkaline phosphatase. The results revealed that the organic LBL template on the PET artificial ligament was effective for CaP apatite formation. Following incubation for 72 h, numerous nanoscale CaP apatites were deposited on the PET ligament fibers. In addition, the results of the in vitro culture of MC3T3-E1 mouse osteoblastic cells demonstrated that the CaP coating had a good biocompatibility for cell proliferation and adhesion, and the CaP-coated group had a significantly higher alkaline phosphatase activity compared with the uncoated control group after seven days of cell culture. Collectively, these results demonstrated that the biomimetic nanoscale CaP-coated PET artificial ligaments have potential in bone-tissue engineering.

Enhanced and suppressed mineralization by acetoacetate and β-hydroxybutyrate in osteoblast cultures

It is known that diabetes aggravates alveolar bone loss associated with periodontitis. While insulin depletion increases the blood concentration of ketone bodies, i.e., acetoacetate and β-hydroxybutyrate, their roles in bone metabolism have not been much studied until today. We investigated the effects of acetoacetate and β-hydroxybutyrate on mineralization of extracellular matrix in cultures of mouse osteoblastic MC3T3-E1 cells and primary mouse osteoblasts in the presence and absence of bone morphogenetic protein-2. Acetoacetate potentiated alkaline phosphatase activity in MC3T3-E1 cells in a concentration-dependent manner, ranging from physiological to pathological concentrations (0.05–5 mmol/L). In contrast, β-hydroxybutyrate lowered it in the same experimental settings. Mineralization in cultures of these cells was also up-regulated by acetoacetate and down-regulated by β-hydroxybutyrate. Similar results were obtained in cultures of mouse primary osteoblasts. Neither alkaline phosphatase mRNA nor its protein expression in MC3T3-E1 cells was affected by acetoacetate or β-hydroxybutyrate, indicating that these ketone bodies control the enzyme activity of alkaline phosphatase in osteoblasts and hence their mineralization bi-directionally. Finally, either gene silencing of monocarboxylate transporter-1, a major transmembrate transporter for ketone bodies, nullified the effects of ketone bodies on alkaline phosphatase activity in MC3T3-E1 cells. Collectively, we found that ketone bodies bidirectionally modulates osteoblast functions, which suggests that ketone bodies are important endogenous factors that regulate bone metabolism in both physiological and pathological situations.

Glucocorticoid Suppresses Connexin 43 Expression by Inhibiting the Akt/mTOR Signaling Pathway in Osteoblasts.

The inhibition of proliferation or functional alteration of osteoblasts by glucocorticoids (GCs) has been recognized as an important etiology of GC-induced osteoporosis (GIO). Connexin 43 (Cx43) is the most abundant connexin isoform in bone cells and plays important roles in bone remodeling. Despite the important role of Cx43 in bone homeostasis and the prevalence of GIO, the direct action of GCs on Cx43 expression in osteoblasts has been poorly described. The aim of the present study was to evaluate how GCs affect Cx43 expression in osteoblasts. Dexamethasone (Dex) treatment decreased expression of Cx43 RNA and protein in MC3T3-E1 mouse osteoblastic cells. Reduction of Cx43 expression by Dex was dependent on the glucocorticoid receptor (GR), as it was abolished by pretreatment with a GR blocker. Treatment with PTH (1-34), a medication used for GIO management, counteracted the suppression of Cx43 by Dex. Akt or mTOR signaling modulators revealed the involvement of the Akt/mTOR signaling pathway in Dex-induced reduction of Cx43 expression. Moreover, overexpression of Cx43 significantly attenuated Dex-inhibited cell viability and proliferation, as evidenced by MTT and bromodeoxyuridine (BrdU) incorporation assay of MC3T3-E1 cells. To account for possible species or cell type differences, human primary osteoblasts were treated with Dex and similar downregulation of Cx43 by Dex was observed. In addition, immunofluorescent staining for Cx43 further demonstrated an apparent decrease in Dex-treated human osteoblasts, while analysis of lucifer yellow propagation revealed reduced gap junction intercellular communication by Dex. Collectively, these findings indicate that GCs suppress Cx43 expression in osteoblasts via GR and the Akt/mTOR signaling pathway and overexpression of Cx43 may, at least in part, rescue osteoblasts from GC-induced reductions in proliferation.

Toll-like receptor-4-dependence of the lipopolysaccharide-mediated inhibition of osteoblast differentiation.

Bone fractures or bones subjected to open conduction and internal fixation are easily infected by bacteria; bacterial lipopolysaccharide (LPS) has been recognized as an important pathogenic factor affecting bone fracture healing. Therefore, the effect of LPS on bone metabolism is relevant for bone healing. In this study, we investigated the effect of LPS on the expression of Toll-like receptor (TLR)-4 (an LPS receptor) by using real-time quantitative PCR and western blotting. We also examined the regulatory role of LPS in osteoblast differentiation by measuring the ALP activity, matrix mineralization, and ALP, OCN, and Runx2 mRNA (essential factors affecting osteoblast differentiation) expression in LPS-treated mouse osteoblast MC3T3-E1 cells. We also evaluated the effect of TLR-4 on LPS-mediated inhibition of osteoblast differentiation using RNA interference. LPS promotes TLR-4 mRNA and protein expression in MC3T3-E1 cells (P < 0.05, P < 0.01 or P < 0.001), and inhibits osteoblast differentiation by downregulating matrix mineralization and ALP activity (P < 0.05, P < 0.01 or P < 0.001), and suppressing the expression ALP, OCN, and Runx2 mRNA in MC3T3-E1 cells (P < 0.05 or P < 0.01). Conversely, RNAi-mediated TLR-4 knockdown abrogates the LPS-mediated inhibition of osteoblast differentiation (P < 0.05 or P < 0.01). In summary, LPS was shown to inhibit osteoblast differentiation by suppressing the expression of ALP, OCN, and Runx2 in a TLR-4-dependent manner. The results of this study may provide insights into the signal pathway of LPS-induced bone loss or delayed bone fracture healing.

Exendin-4 promotes proliferation and differentiation of MC3T3-E1 osteoblasts by MAPKs activation.

Glucagon-like peptide-1 (GLP1) and its receptor agonist have been previously reported to play a positive role in bone metabolism in aged ovariectomized rats and insulin-resistant models. However, whether GLP1 has a direct effect on the proliferation and differentiation of osteoblasts or any cellular mechanism for this potential role is unknown. We examined the effects of the GLP1 receptor agonist exendin-4 on the proliferation, differentiation, and mineralization of mouse osteoblastic MC3T3-E1 cells. GLP1 receptor was detected in MC3T3-E1 cells by polymerase chain reaction (PCR) and Western blot assay. Cell proliferation was assessed using MTT assay, revealing that exendin-4 increased cell proliferation at effective concentrations between 10(-10) and 10(-5) M. Quantitative PCR analysis showed that exendin-4 increased the mRNA expression of the differentiation markers alkaline phosphatase (ALP), collagen-1 (COL1), osteocalcin (OC), and runt-related transcription factor 2 (RUNX2) under osteogenic conditions. Alizarin red staining confirmed that 10(-7) M exendin-4 increased osteoblast mineralization by 18.7%. Exendin-4 upregulated the phosphorylation of ERK1/2, p38, and JNK, with the peak effect at 1.5 h in the Western blot analysis. The use of selective MAPK inhibitors, namely PD98059, SB203580, and SP600125, blocked the effects of exendin-4 on kinase activation (ERK1/2, p38, and JNK), as well as cell proliferation and differentiation in MC3T3-E1 cells. These findings demonstrate that exendin-4 promotes both the proliferation and differentiation of preosteoblasts MC3T3-E1 via activation of the MAPK pathway.

The Transcription Factor EB (TFEB) Regulates Osteoblast Differentiation Through ATF4/CHOP-Dependent Pathway.

Osteoblasts are bone-forming cells that produce large amounts of collagen type I and various bone matrix proteins. Although osteoblast differentiation is highly regulated by various factors, it remains unknown whether lysosomes are directly involved in osteoblast differentiation. Here, we demonstrate the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, modulates osteoblast differentiation. The expression levels of TFEB as well as those of endosomal/lysosomal proteins were up-regulated during osteoblast differentiation using mouse osteoblastic MC3T3-E1 cells. By gene knockdown (KD) experiments with small interfering RNA (siRNA), TFEB depletion caused markedly reduced osteoblast differentiation as compared with the control cells. Conversely, overexpression (OE) of TFEB resulted in strikingly enhanced osteoblastogenesis compared to the control cells. By analysis of down-stream effector molecules, TFEB KD was found to cause marked up-regulation of activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP), both of which are essential factors for osteoblastogenesis. In contrast, TFEB OE promoted osteoblast differentiation through reduced expression of ATF4 and CHOP without differentiation agents. Given the importance of ATF4 and CHOP in osteoblastogenesis, it is clear that the TFEB-regulated signaling pathway for osteoblast differentiation is involved in ATF4/CHOP-dependent signaling pathway.

Resveratrol inhibits the hydrogen dioxide-induced apoptosis via Sirt 1 activation in osteoblast cells.

Sirt 1 plays a critical role in stress responses. We determined the deregulation of Sirt 1 activity, p53 acetylation, Bcl-2 expression, and mitochondria-dependent apoptosis in mouse osteoblast MC3T3-E1 cells which were exposed to H2O2. And then we investigated the protective role of Sirt 1 activator, Resveratrol (RSV), against the H2O2-induced apoptosis. Results demonstrated that Sirt 1 and Bcl-2 were inhibited, whereas p53 acetylation, Bax, and caspase 9 were promoted by H2O2, as was aggravated by the Sirt 1 inhibitor, EX-527. Instead, RSV inhibited the H2O2-induced both p53 acetylation and the caspase 9 activation, whereas ameliorated the H2O2-induced Bcl-2 inhibition and apoptosis. In conclusion, Sirt 1 was downregulated during the H2O2-induced apoptosis in MC3T3-E1 cells. And the chemical activation of Sirt 1 inhibited the H2O2-induced apoptosis via the downregulation of p53 acetylation. Our results suggest that Sirt 1 upregulation appears to be an important strategy to inhibit the oxidative stress-induced apoptosis.