Osteoblast Differentiation In Mice Research Articles

Potential osteoporosis-blocker Sparassis crispa polysaccharide: Isolation, purification and structure elucidation

This study aimed to investigate the structural characterization of water-soluble polysaccharides from Sparassis crispa and their effects on the proliferation and differentiation of mouse osteoblasts. Three fractions (F-1, F-2, and F-3) were obtained from crude polysaccharides by a DEAE-52 cellulose column. The main fraction (F-1) was further purified by polysaccharide gel purification systems to obtain purified water-soluble Sparassis crispa polysaccharide (SCPS). The chemical structure of SCPS was analyzed by gas chromatography, Fourier transform infrared spectroscopy, methylation analysis, and nuclear magnetic resonance spectroscopy. The monosaccharide compositional analysis revealed that SCPS consisted of fucose, arabinose, galactose, glucose, xylose, mannose, ribose, galacturonic acid, glucuronic acid, and mannuronic acid in a molar ratio of 17.37:1.94:25.52:30.83:1.14:0.30:4.98:2.87:2.65. Moreover, the backbone of SCPS was composed of →3)-β-d-Glcp-(1→4)-β-d-Glcp-(1→, with side chains attached to the backbone at the O-6 positions through the →3,6)-β-d-Glcp-(1→ linkage. The in vitro experiments were conducted to investigate the effects of SCPS on the proliferation and differentiation of mouse osteoblasts. The results showed that SCPS significantly enhanced the proliferation and differentiation of mouse osteoblasts, indicating their potential as a pharmaceutical agent for promoting osteoblast proliferation and differentiation.

Electron beam radiation constructed and improved bone repairing performance based on hierarchical thermoplastic polyurethane/graphene oxide/hydroxyapatite hot melt adhesive with high flow ductility

AbstractThe aging population increased the demand for the development and utilization of bone filling materials. In this study, hydroxyapatite (HAP) and graphene oxide (GO) were introduced into thermoplastic polyurethane (TPU) by melt mixing and electron beam radiation to obtain the new hot melt adhesive (TPU/HAP/GO). The structures and morphologies of the TPU/HAP/GO were analyzed using FTIR, XRD, SEM, BET, DSC. The mechanical properties of the TPU/HAP/GO were evaluated using a universal testing machine. TPU/HAP/GO was demonstrated to have good bacterial inhibition by the zone of inhibition test and plate method. The good biocompatibility of the hot melt adhesive was demonstrated using the MTT method and the Hoechst 33342/PI double staining method. The osteogenic differentiation of mouse osteoblasts (MC3T3‐E1) treated with TPU/HAP/GO was detected by alkaline phosphatase (ALP) staining. ALP staining showed that the hot melt adhesive promoted the differentiation of MC3T3‐E1 cells into osteoblasts. This hot melt adhesive had great application potential in bone tissue engineering.Highlights Green electron beam radiation method for synthesizing hot melt adhesives. Enhanced mechanical properties of composites by incorporation of graphene oxide. Improvement of antimicrobial and osteogenic capacities for polyurethane.

Acyl-protein thioesterase1 alleviates senile osteoporosis by promoting osteoblast differentiation via depalmitoylation of BMPR1a

ObjectiveSenile osteoporosis (SOP) is an aging-related disease. The depalmitoylating enzyme Acyl-protein thiesterase1 (APT1) is involved in disease regulation. This study explored the mechanism of APT1 in SOP. MethodsEight-week-old SAMP6 mice were selected as SOP models and SAMR1 mice were controls, while osteoblasts were isolated from the femoral surface-soft tissues of SOP and control mice as in vitro models. Mouse femur morphological, bone mineral density (BMD), femur maximum elastic stress and maximum load, and APT1 expression were detected by HE staining, X-ray bone densitometer, material testing machine, and RT-qPCR and Western blot (WB). Osteoprotegrin (OPG)-labeled osteoblasts and APT1 localization in bone tissues were detected by immunohistochemical staining. APT1 expression was promoted in SOP mice by tail vein injection of APT1 lentivirus or promoted/silenced in osteoblasts by transfection of pcDNA3.1-APT1 overexpression or si-APT1 plasmids. SOP mouse osteoblast differentiation (OD), OD-related protein levels, osteoblast proliferation, BMPR1a palmitoylation level, and BMP/Smad pathway were detected by alizarin red staining, ALP activity detection, WB, CCK-8, and IP-ABE method. The effects of the pathway inhibitor LDN-193189 on OD were detected. ResultsAPT1 was under-expressed in osteoblasts of bone tissue in SOP mice and mainly localized in osteoblasts. SOP mice manifested increased bone marrow cavity and bone trabecular space, thinned trabecular bone, decreased BMD, maximum elastic stress, maximum load, and reduced OPG-positive osteoblasts in bone tissues, which were averted by APT1 overexpression, thus alleviating SOP. APT1 overexpression increased osteoblast calcium nodules, ALP activity, OD-related protein levels, and cell proliferation. In mechanism, APT1 overexpression inhibited BMPR1a palmitoylation in SOP mouse osteoblasts and activated the BMP/Smad pathway, thus promoting OD. ConclusionAPT1 activated the BMP/Smad pathway and promoted OD by regulating BMPR1a depalmitoylation, thus alleviating mouse SOP.

Myricitrin promotes osteogenesis and prevents ovariectomy bone mass loss via the PI3K/AKT signalling pathway.

This study aimed to explore the effect of myricitrin on osteoblast differentiation in mice immortalised bone marrow mesenchymal stem cells (imBMSCs). Additionally, ovariectomy (OVX) mice were employed to examine the effect of myricitrin on bone trabecular loss in vivo. The effect of myricitrin on the proliferation of imBMSCs was evaluated using a cell counting kit-8 assay. Alizarin red staining, alkaline phosphatase staining were performed to elucidate osteogenesis. Furthermore, qRT-PCR and western blot determined the expression of osteo-specific genes and proteins. To screen for candidate targets, mRNA transcriptome genes were sequenced using bioinformatics analyses. Western blot and molecular docking analysis were used to examine target signalling markers. Moreover, rescue experiments were used to confirm the effect of myricitrin on the osteogenic differentiation of imBMSCs. OVX mice were also used to estimate the delay capability of myricitrin on bone trabecular loss in vivo using western blot, micro-CT, tartaric acid phosphatase (Trap) staining, haematoxylin and eosin staining, Masson staining and immunochemistry. In vitro, myricitrin significantly enhanced osteo-specific genes and protein expression and calcium deposition. Moreover, mRNA transcriptome gene sequencing and molecular docking analysis revealed that this enhancement was accompanied by an upregulation of the PI3K/AKT signalling pathway. Furthermore, copanlisib, a PI3K inhibitor, partially reversed the osteogenesis promotion induced by myricitrin. In vivo, western blot, micro-CT, hematoxylin and eosin staining, Masson staining, Trap staining and immunochemistry revealed that bone trabecular loss rate was significantly alleviated in the myricitrin low- and high-dose groups, with an increased expression of osteopontin, osteoprotegerin, p-PI3K and p-AKT compared to the OVX group. Myricitrin enhances imBMSC osteoblast differentiation and attenuate bone mass loss partly through the upregulation of the PI3K/AKT signalling pathway. Thus, myricitrin has therapeutic potential as an antiosteoporosis drug.

AS-605240 Blunts Osteoporosis by Inhibition of Bone Resorption.

Osteoporosis is a metabolic bone disease. Osteoclasts are significantly involved in the pathogenesis of osteoporosis. AS-605240 (AS) is a small molecule PI3K-γ inhibitor and is less toxic compared to pan-PI3K inhibitors. AS also exerts multiple biological effects including anti-inflammatory, anti-tumor, and myocardial remodeling promotion. However, the involvement of AS in the differentiation and functions of osteoclasts and the effect of AS in treating patients with osteoporosis is still unclear. This study aimed to investigate if AS inhibits the differentiation of osteoclasts and resorption of the bones induced by M-CSF and RANKL. Next, we evaluated the therapeutic effects of AS on bone loss in ovariectomy (OVX)-induced osteoporosis mice models. We stimulated bone marrow-derived macrophages with an osteoclast differentiation medium containing different AS concentrations for 6 days or 5μM AS at different times. Next, we performed tartrate-resistant acid phosphatase (TRAP) staining, bone resorption assay, F-actin ring fluorescence, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blotting (WB). Next, MC3T3-E1s (pre-osteoblast cells) were differentiated to osteoblast by stimulating the cells with varying AS concentrations. Next, we performed alkaline phosphatase (ALP) staining, RT-qPCR, and WB on these cells. We established an OVX-induced osteoporosis mice model and treated the mice with 20mg/kg of AS. Finally, we extracted the femurs and performed micro-CT scanning, H&E, and TRAP staining. AS inhibits the formation of osteoclasts and resorption of bone triggered by RANKL by inhibiting the PI3K/Akt signaling pathway. Furthermore, AS enhances the differentiation of osteoblasts and inhibits bone loss due to OVX in vivo. AS inhibits osteoclast production and enhances osteoblast differentiation in mice, thus providing a new therapeutic approach for treating patients with osteoporosis.

Meta-Analysis of Mechanism of Influence of CRY2 on the Differentiation of Mouse Osteoblast through the Regulation of Wnt/Β-Catenin Signaling Pathway.

Noncoding RNAs were discovered to control a variety of developmental mechanisms, including osteogenesis. According to emerging evidence, cryptochrome circadian-regulating (CRY) proteins have emerged as essential controllers of osteoblast differentiation. The linked processes, on the other hand, are still unknown. The specific process that underpins osteoblast differentiation and proliferation is yet unknown. This research gives a meta-analysis of CRY2's impact on mouse osteoblast differentiation via the control of the WNT/β–catenin signaling pathways. Western blot and quantitative real-time PCR were used to identify Cry2 expression levels, components in the osteoblast-associated signaling pathway, and osteoblast transcription markers. The osteogenic condition was measured utilizing alkaline phosphatase (ALP) and alizarin red (AR) staining, whereas cell growth rates were measured using CCK8 assays. An ectopic bone formation experiment was used to determine the osteogenic potential of osteoblasts. Cry2 stimulates the osteogenic development of mouse osteoblasts through canonical Wnt/β-catenin signaling, according to the findings.

Involvement of miRNA mediated-upregulation of Kir2.1 in mouse osteoblast differentiation

Involvement of miRNA mediated-upregulation of Kir2.1 in mouse osteoblast differentiation

MYOC Promotes the Differentiation of C2C12 Cells by Regulation of the TGF-β Signaling Pathways via CAV1.

Simple SummaryMYOC is a secreted glycoprotein and it expresses at high levels in skeletal muscle cells. However, the function of MYOC in muscle is still unclear. Accordingly, in this study, we examined that MYOC expression increased gradually during C2C12 differentiation and it could promote the differentiation of C2C12. Furthermore, we demonstrated that MYOC could bind to CAV1. We further confirmed that CAV1 could positively regulate C2C12 differentiation through the TGF-β pathway. At last, we determined the relationship among MYOC, CAV1 and TGF-β. We found that MYOC promoted the differentiation of C2C12 cells by regulation of the TGF-β signaling pathways via CAV1. The present study is the first to demonstrate the mechanism of action of MYOC in C2C12 cells. It provides a novel method of exploring the mechanism of muscle differentiation and represents a potential novel method for the treatment of muscle diseases.Myocilin (MYOC) is a glycoprotein encoded by a gene associated with glaucoma pathology. In addition to the eyes, it also expresses at high transcription levels in the heart and skeletal muscle. MYOC affects the formation of the murine gastrocnemius muscle and is associated with the differentiation of mouse osteoblasts, but its role in the differentiation of C2C12 cells has not yet been reported. Here, MYOC expression was found to increase gradually during the differentiation of C2C12 cells. Overexpression of MYOC resulted in enhanced differentiation of C2C12 cells while its inhibition caused reduced differentiation. Furthermore, immunoprecipitation indicated that MYOC binds to Caveolin-1 (CAV1), a protein that influences the TGF-β pathway. Laser confocal microscopy also revealed the common sites of action of the two during the differentiation of C2C12 cells. Additionally, CAV1 was upregulated significantly as C2C12 cells differentiated, with CAV1 able to influence the differentiation of the cells. Furthermore, the Western blotting analysis demonstrated that the expression of MYOC affected the TGF-β pathway. Finally, MYOC was overexpressed while CAV1 was inhibited. The results indicate that reduced CAV1 expression blocked the promotion of C2C12 cell differentiation by MYOC. In conclusion, the results demonstrated that MYOC regulates TGF-β by influencing CAV1 to promote the differentiation of C2C12 cells.

Effects of inhibiting PDK‑1 expression in bone marrow mesenchymal stem cells on osteoblast differentiation invitro.

Osteoblasts are the main functional cells in bone formation, which are responsible for the synthesis, secretion and mineralization of bone matrix. The PI3K/AKT signaling pathway is strongly associated with the differentiation and survival of osteoblasts. The 3-phosphoinositide-dependent protein kinase-1 (PDK-1) protein is considered the master upstream lipid kinase of the PI3K/AKT cascade. The present study aimed to investigate the role of PDK-1 in the process of mouse osteoblast differentiation in vitro. In the BX-912 group, BX-912, a specific inhibitor of PDK-1, was added to osteoblast induction medium (OBM) to treat bone marrow mesenchymal stem cells (BMSCs), whereas the control group was treated with OBM alone. Homozygote PDK1flox/flox mice were designed and generated, and were used to obtain BMSCsPDK1flox/flox. Subsequently, an adenovirus containing Cre recombinase enzyme (pHBAd-cre-EGFP) was used to disrupt the PDK-1 gene in BMSCsPDK1flox/flox; this served as the pHBAd-cre-EGFP group and the efficiency of the disruption was verified. Western blot analysis demonstrated that the protein expression levels of phosphorylated (p)-PDK1 and p-AKT were gradually increased during the osteoblast differentiation process. Notably, BX-912 treatment and disruption of the PDK-1 gene with pHBAd-cre-EGFP effectively reduced the number of alkaline phosphatase (ALP)-positive cells and the optical density value of ALP activity, as well as the formation of cell mineralization. The mRNA expression levels of PDK-1 in the pHBAd-cre-EGFP group were significantly downregulated compared with those in the empty vector virus group on days 3–7. The mRNA expression levels of the osteoblast-related genes RUNX2, osteocalcin and collagen I were significantly decreased in the BX-912 and pHBAd-cre-EGFP groups on days 7 and 21 compared with those in the control and empty vector virus groups. Overall, the results indicated that BX-912 and disruption of the PDK-1 gene in vitro significantly inhibited the differentiation and maturation of osteoblasts. These experimental results provided an experimental and theoretical basis for the role of PDK-1 in osteoblasts.

An in situ hybridization study of decorin and biglycan mRNA in mouse osteoblasts in vivo.

In situ hybridization of decorin and biglycan mRNA, principal members of small leucine-rich proteoglycan, was performed using [35S]-labeled RNA probes, in the context of the hypothesis that they show different expression patterns associated with osteoblast differentiation in mice. We adopted two ossifying sites that can clearly follow the developmental process of bone formation: ossifying tympanic ring and developing bone collar of mandibular condylar cartilage. Decorin mRNA was expressed in osteoblasts of developing tympanic ring at E14.0, as well as of developing bone collar at E15.0, but biglycan mRNA was not, indicating decorin mRNA was expressed earlier in newly differentiating osteoblasts than biglycan. With maturation of osteoblasts, biglycan mRNA became expressed and maintained its expression both in the outer region (periosteum) and in the interior region (endosteum) of bone. By contrast, decorin mRNA expression was maintained in the outer region but diminished in the interior region. These results indicate that decorin and biglycan show differential expression patterns in differentiating osteoblasts and play specific roles in bone formation.

Glycine Lipids of Porphyromonas gingivalis Are Agonists for Toll-Like Receptor 2.

The serine-glycine dipeptide lipid classes, including lipid 430 and lipid 654, are produced by the periodontal pathogen Porphyromonas gingivalis and can be detected in lipid extracts of diseased periodontal tissues and teeth of humans. Both serine-glycine lipid classes were previously shown to engage human and mouse Toll-like receptor 2 (TLR2) and to inhibit mouse osteoblast differentiation and function through engagement of TLR2. It is not clear if other lipids related to serine-glycine lipids are also produced by P. gingivalis The goal of this investigation was to determine whether P. gingivalis produces additional lipid classes similar to the serine-glycine lipids that possess biological properties. P. gingivalis (ATCC 33277) was grown in broth culture, and lipids were extracted and fractionated by high-performance liquid chromatography (HPLC). Lipids were separated using semipreparative HPLC, and specific lipid classes were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometric approaches. Two glycine lipid classes were identified, termed lipid 567 and lipid 342, and these lipid classes are structurally related to the serine-glycine dipeptide lipids. Both glycine lipid classes were shown to promote TLR2-dependent tumor necrosis factor alpha (TNF-α) release from bone marrow macrophages, and both were shown to activate human embryonic kidney (HEK) cells through TLR2 and TLR6 but not TLR1. These results demonstrate that P. gingivalis synthesizes glycine lipids and that these lipids engage TLR2 similarly to the previously reported serine-glycine dipeptide lipids.

A novel long noncoding RNA PGC1β-OT1 regulates adipocyte and osteoblast differentiation through antagonizing miR-148a-3p.

Long noncoding RNAs (LncRNAs) have been implicated in the regulation of adipocyte and osteoblast differentiation. However, the functional contributions of LncRNAs to adipocyte or osteoblast differentiation remain largely unexplored. In the current study we have identified a novel LncRNA named peroxisome proliferator-activated receptor γ coactivator-1β-OT1 (PGC1β-OT1). The expression levels of PGC1β-OT1 were altered during adipogenic and osteogenic differentiation from progenitor cells. 5'- and 3'-rapid amplification of cDNA ends (RACE) revealed that PGC1β-OT1 is 1759 nt in full length. Overexpression of PGC1β-OT1 in progenitor cells inhibited adipogenic differentiation, whereas silencing of endogenous PGC1β-OT1 induced adipogenic differentiation. By contrast, overexpression of PGC1β-OT1 in progenitor cells stimulated, whereas silencing of PGC1β-OT1 inhibited osteogenic differentiation. In vivo experiment showed that silencing of endogenous PGC1β-OT1 in marrow stimulated fat accumulation and decreased osteoblast differentiation in mice. Mechanism investigations revealed that PGC1β-OT1 contains a functional miR-148a-3p binding site. Overexpression of the mutant PGC1β-OT1 with mutation at the binding site failed to regulate either adipogenic or osteogenic differentiation. In vivo crosslinking combined with affinity purification studies demonstrated that PGC1β-OT1 physically associated with miR-148a-3p through the functional miR-148a-3p binding site. Furthermore, PGC1β-OT1 affected the expression of endogenous miR-148a-3p and its target gene lysine-specific demethylase 6b (KDM6B). Supplementation of miR-148a-3p in progenitor cells blocked the inhibitory effect of PGC1β-OT1 on adipocyte formation. Moreover, overexpression of Kdm6b restored the osteoblast differentiation which was inhibited by silencing of endogenous PGC1β-OT. Our studies provide evidences that the novel LncRNA PGC1β-OT1 reciprocally regulates adipogenic and osteogenic differentiation through antagonizing miR-148a-3p and enhancing KDM6B effect.

Regulator of G Protein Signaling Protein 12 (Rgs12) Controls Mouse Osteoblast Differentiation via Calcium Channel/Oscillation and Gαi-ERK Signaling.

Bone homeostasis intimately relies on the balance between osteoblasts (OBs) and osteoclasts (OCs). Our previous studies have revealed that regulator of G protein signaling protein 12 (Rgs12), the largest protein in the Rgs super family, is essential for osteoclastogenesis from hematopoietic cells and OC precursors. However, how Rgs12 regulates OB differentiation and function is still unknown. To understand that, we generated an OB-targeted Rgs12 conditional knockout (CKO) mice model by crossing Rgs12fl/fl mice with Osterix (Osx)-Cre transgenic mice. We found that Rgs12 was highly expressed in both OB precursor cells (OPCs) and OBs of wild-type (WT) mice, and gradually increased during OB differentiation, whereas Rgs12-CKO mice (OsxCre/+ ; Rgs12fl/fl ) exhibited a dramatic decrease in both trabecular and cortical bone mass, with reduced numbers of OBs and increased apoptotic cell population. Loss of Rgs12 in OPCs in vitro significantly inhibited OB differentiation and the expression of OB marker genes, resulting in suppression of OB maturation and mineralization. Further mechanism study showed that deletion of Rgs12 in OPCs significantly inhibited guanosine triphosphatase (GTPase) activity and cyclic adenosine monophosphate (cAMP) level, and impaired Calcium (Ca2+ ) oscillations via restraints of major Ca2+ entry sources (extracellular Ca2+ influx and intracellular Ca2+ release from endoplasmic reticulum), partially contributed by the blockage of L-type Ca2+ channel mediated Ca2+ influx. Downstream mediator extracellular signal-related protein kinase (ERK) was found inactive in OBs of OsxCre/+ ; Rgs12fl/fl mice and in OPCs after Rgs12 deletion, whereas application of pertussis toxin (PTX) or overexpression of Rgs12 could rescue the defective OB differentiation via restoration of ERK phosphorylation. Our findings reveal that Rgs12 is an important regulator during osteogenesis and highlight Rgs12 as a potential therapeutic target for bone disorders. © 2018 American Society for Bone and Mineral Research.

Review: Novel Insights Into Tumor Necrosis Factor Receptor, Death Receptor 3, and Progranulin Pathways in Arthritis and Bone Remodeling.

Approximately 30 members of the tumor necrosis factor receptor superfamily (TNFRSF) have been identified. They are transmembrane proteins with cysteine‐rich motifs in their extracellular domains that bind to their cognate ligands 1. They are categorized into 3 groups: death domain–containing receptors, decoy receptors, and TNFR‐associated factor–binding receptors. Only 8 TNFRSF members contain a death domain (TNFR type I [TNFRI], death receptor 3 [DR‐3], DR‐4, DR‐5, DR‐6, Fas, nerve growth factor receptor, and ectodysplasin A receptor [EDAR]), of which TNFRI and DR‐3 constitute the principal focus of this article. Interactions between TNF superfamily (TNFSF) ligands and TNFRSF receptors help maintain tissue homeostasis by controlling survival, proliferation, differentiation, and effector function of immune cells. We limit our review to recent advances and novel insights into the roles of TNFRI and DR‐3 in bone and joint biology. Bone cells (osteoblasts, osteoclasts, and osteocytes), fibroblast‐like synoviocytes, chondrocytes, and immune cells that infiltrate the arthritic joint will at different times express a wide range of TNFRSF members and TNFSF ligands. An overview of the current status of our knowledge in this regard is provided in Table 1. The impact of TNFRI activation on bone and inflammatory joint diseases has been researched in great depth 2, 3, but little or no data in the field have been reported on other more recently discovered TNFRSF members such as TROY (TNFRSF expressed on the mouse embryo; TNFRSF19), EDAR, and XEDAR (X‐linked ectodysplasin receptor; TNFRSF27). The unexpected interaction between progranulin (PGRN) and both TNFRI and TNFRII is particularly interesting in the context of arthritis‐associated bone pathology. PGRN levels are elevated in the synovial fluid of patients with rheumatoid arthritis (RA), osteoarthritis (OA), and other arthropathies 4, 5, 6, and PGRN has been shown to inhibit TNF‐induced osteoclastogenesis and promote osteoblast differentiation in mice 7. However, PGRN has a higher binding affinity for TNFRII (antiinflammatory with osteoprotective function) than for TNFRI (predominantly proinflammatory with degenerative function), which suggests conflicting actions. The potential overall impact of these divergent PGRN signaling pathways on the architecture of the arthritic joint has been evaluated 8. Table 1 Cellular expression of death domain–containing TNFRSF members and their association with arthritis*

Histone demethylase JMJD3 is required for osteoblast differentiation in mice.

JMJD3 (KDM6B) is an H3K27me3 demethylases and emerges as an important player in developmental processes. Although some evidence indicated the involvement of JMJD3 in osteoblast differentiation in vitro, its role as a whole in osteoblast differentiation and bone formation in vivo remains unknown. Here we showed that homozygous deletion of Jmjd3 resulted in severe delay of osteoblast differentiation and bone ossification in mice. By biochemical and genetical methods, we demonstrated that JMJD3 mediated RUNX2 transcriptional activity and cooperated with RUNX2 to promote osteoblast differentiation and bone formation in vivo. These results strongly demonstrated that JMJD3 is required for osteoblast differentiation and bone formation in mice.

DNA–PKcs–SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation

Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA–PKcs)–Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which was detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1Gy) induced phosphorylation of DNA–PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA–PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA–PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA–PKcs–SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA–PKcs–SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.

Effects of X-ray doses on mouse osteoblasts in vitro

Objective To investigate the effects of different X-ray doses on the proliferation and differentiation of mouse osteoblasts in vitro.Methods The mouse osteoblastic-like cell line (MC3T3-E1)was cultured in vitro and divided into 4 groups (n =3).On the second day after seeding,the cells in the 4 groups (control group,0.1 Gy group,0.5 Gy group and 1.0 Gy group) were exposed to X-ray irradiation respectively at 4 different doses of 0,0.1,0.5,1.0 Gy.Cell proliferation,cell cycle and apoptosis were measured on day 1 to day 3 after irradiation; alkaline phosphatase (ALP) activity on days 7,10 and 14 and mineralization on day 14 were evaluated respectively.The mRNA expression of osteocalcin (OCN),core-binding factor α1 (Cbfα1),osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B lig-and (RANKL) were measured by real time-PCR on day 14.Results Increasing doses of X-ray irradiation had no significant effects on cell proliferation,cell cycle or apoptosis compared with the control group(P ＞0.05).Importantly,X-ray irradiation of 0.5 Gy and 1.0 Gy increased ALP activities and mineralized nodules of osteoblasts,and markedly increased mRNA expressions of OPG/RANKL (P ＜ 0.05).Specially,mRNA expressions of OCN and Cbfα1 were also markedly increased after X-ray irradiation of 1.0 Gy (P ＜ 0.05).Conclusion X-ray irradiation at a certain rage of doses (from 0.5 to 1.0 Gy) can promote differentiation and mineralization of mouse osteoblasts but does not affect their proliferation and apoptosis. Key words: X-rays; Osteoblasts; Cell proliferation; Cell differentiation

MiR-145 and miR-143 Regulate Odontoblast Differentiation through Targeting Klf4 and Osx Genes in a Feedback Loop

Dentin tissue is derived from mesenchymal cells induced into the odontoblast lineage. The differentiation of odontoblasts is a complex process regulated by several transcriptional factor signaling transduction pathways. However, post-translational regulation of these factors during dentinogenesis remains unclear. To further explore the mechanisms, we investigated the role of microRNA (miRNA) during odontoblast differentiation. We profiled the miRNA expression pattern during mouse odontoblast differentiation using a microarray assay and identified that miR-145 and miR-143 were down-regulated during this process. In situ hybridization verified that the two miRNAs were gradually decreased during mouse odontoblast differentiation. Loss-of-function and gain-of-function experiments revealed that down-regulation of miR-145 and miR-143 could promote odontoblast differentiation and increased Dspp and Dmp1 expression in mouse primary dental pulp cells and vice versa. We found that miR-145 and miR-143 controlled odontoblast differentiation through several mechanisms. First, KLF4 and OSX bind to their motifs in Dspp and Dmp1 gene promoters and up-regulate their transcription thereby inducing odontoblast differentiation. The miR-145 binds to the 3'-UTRs of Klf4 and Osx genes, inhibiting their expression. Second, KLF4 repressed miR-143 transcription by binding to its motifs in miR-143 regulatory regions as detected by ChIP assay and dual luciferase reporter assay. Third, miR-143 regulates odontoblast differentiation in part through miR-145 pathway. Taken together, we for the first time showed that the miR-143 and miR-145 controlled odontoblast differentiation and dentin formation through KLF4 and OSX transcriptional factor signaling pathways.

Gsα enhances commitment of mesenchymal progenitors to the osteoblast lineage but restrains osteoblast differentiation in mice

The heterotrimeric G protein subunit Gsα stimulates cAMP-dependent signaling downstream of G protein-coupled receptors. In this study, we set out to determine the role of Gsα signaling in cells of the early osteoblast lineage in vivo by conditionally deleting Gsα from osterix-expressing cells. This led to severe osteoporosis with fractures at birth, a phenotype that was found to be the consequence of impaired bone formation rather than increased resorption. Osteoblast number was markedly decreased and osteogenic differentiation was accelerated, resulting in the formation of woven bone. Rapid differentiation of mature osteoblasts into matrix-embedded osteocytes likely contributed to depletion of the osteoblast pool. In addition, the number of committed osteoblast progenitors was diminished in both bone marrow stromal cells (BMSCs) and calvarial cells of mutant mice. In the absence of Gsα, expression of sclerostin and dickkopf1 (Dkk1), inhibitors of canonical Wnt signaling, was markedly increased; this was accompanied by reduced Wnt signaling in the osteoblast lineage. In summary, we have shown that Gsα regulates bone formation by at least two distinct mechanisms: facilitating the commitment of mesenchymal progenitors to the osteoblast lineage in association with enhanced Wnt signaling; and restraining the differentiation of committed osteoblasts to enable production of bone of optimal mass, quality, and strength.

Ginsenoside Rg1, a Novel Glucocorticoid Receptor Agonist of Plant Origin, Maintains Glucocorticoid Efficacy with Reduced Side Effects

Glucocorticoids (GCs) are widely used to treat inflammatory diseases. However, they cause debilitating side effects, which limit the use of these compounds. In the past decade, many researchers have attempted to find so-called dissociated GCs that have separate distinct transactivation and transrepression activities. Anti-inflammation of GCs is a result of glucocorticoid receptor (GR)-mediated transactivation and transrepression in some tissues, similar to their side effects; therefore, the goal to discover a compound that has anti-inflammatory properties, but lacks the negative side effects seen with GCs, has yet to be achieved. In the present study, we introduce a plant-derived compound, ginsenoside Rg1, which possesses GC and estrogen-like activities. In this study, we show that Rg1 downmodulates LPS-induced proinflammatory cytokine release and inhibits NF-κB nuclear translocation and DNA binding activity. The negative effects on NF-κB activation are due to a decrease in IκB phosphorylation and protein stabilization. Furthermore, the inhibitory effect of Rg1 on NF-κB is GR-dependent, as small interfering RNA knockdown of GR abrogated this function. Rg1 also displayed profound inhibitory effects on LPS-induced MAPK activation. Importantly, Rg1 did not impair proliferation or differentiation of mouse osteoblasts. Finally, we show that Rg1 can effectively inhibit acute and chronic inflammation in vivo, but it does not cause hyperglycemia or osteoporosis as seen with dexamethasone. These results suggest that ginsenoside Rg1 may serve as a novel anti-inflammatory agent and may exhibit a potential profile for therapeutic intervention in inflammatory diseases.