Osteoblast-like MC3T3-E1 Cells Research Articles

Oncostatin M suppresses bone morphogenetic protein-4-induced osteoprotegerin synthesis in MC3T3-E1 osteoblast-like cells: p70 S6 kinase attenuation.

Evidence is accumulating that osteal macrophages, in addition to bone-resorbing osteoclasts and bone-forming osteoblasts, participate vitally in bone remodeling process. Oncostatin M (OSM), an inflammatory cytokine belonging to interleukin-6 superfamily, is recognized as an essential factor secreted by osteal macrophages to orchestrate bone remodeling. Osteoprotegerin (OPG) produced by osteoblasts regulates osteoclastogenesis. We have reported that bone morphogenetic protein-4 (BMP-4) stimulates OPG synthesis in MC3T3-E1 osteoblast-like cells, and that SMAD1/5/8(9), p38 mitogen-activated protein kinase (MAPK), and p70 S6 kinase are involved in the OPG synthesis. The present study aims to investigate the effect of OSM on the synthesis of OPG stimulated by BMP-4 in osteoblasts. OSM suppressed the release and the mRNA expression of OPG upregulated by BMP-4 in MC3T3-E1 cells. Neither the BMP-4-induced phosphorylation of SMAD1/5/9 nor that of p38 MAPK was affected by OSM. On the other hand, the phosphorylation of p70 S6 kinase stimulated by BMP-4 was considerably suppressed by OSM. These results strongly suggest that OSM suppresses the BMP-4-stimulated OPG synthesis via inhibition of the p70 S6 kinase-mediated pathway in osteoblast-like cells.

Gallein increases the fibroblast growth factor 2-elicited osteoprotegerin synthesis in osteoblasts

Gallein is known as an inhibitor of Gβγ subunits, but roles of gallein in bone metabolism have not been reported. Fibroblast growth factor 2 (FGF-2) increases angiogenesis and promotes bone regeneration during the early stages of fracture healing. Osteoprotegerin (OPG) secreted by osteoblasts, binds to the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) as a decoy receptor and prevents RANKL from binding to RANK, resulting in the suppression of bone resorption. Our previous report demonstrated that FGF-2 activates the phosphorylation of p38 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (JNK), and p44/p42 MAPK in osteoblast-like MC3T3-E1 cells. Additionally, FGF-2-activated phosphorylation of p38 MAPK and JNK but not p44/p42 MAPK is positively involved in OPG synthesis in these cells. This work aimed to investigate the effects of gallein on the FGF-2-elicited OPG synthesis in osteoblast-like MC3T3-E1 cells and the mechanism. Our findings demonstrated that gallein significantly increased the FGF-2-elicited OPG synthesis in MC3T3-E1 cells. By contrast, fluorescein, gallein-like compound that does not bind Gβγ, did not affect the FGF-2-elicited OPG synthesis. Gallein significantly enhanced the FGF-2-induced OPG mRNA expression levels. Gallein did not affect the FGF-2-activated phosphorylation of p38 MAPK and p44/p42 MAPK, but significantly increased the FGF-2-activated phosphorylation of JNK, while fluorescein did not affect JNK phosphorylation. SP600125, a specific JNK inhibitor, strongly inhibited gallein-induced enhancement of FGF-2-induced OPG synthesis and mRNA expression levels. Our results indicated that gallein increases the FGF-2-induced OPG synthesis due to the JNK activation in the osteoblast.

Gprc5a is a novel parathyroid hormone-inducible gene and negatively regulates osteoblast proliferation and differentiation.

Teriparatide is a peptide derived from a parathyroid hormone (PTH) and an osteoporosis therapeutic drug with potent bone formation-promoting activity. To identify novel druggable genes that act downstream of PTH signaling and are potentially involved in bone formation, we screened PTH target genes in mouse osteoblast-like MC3T3-E1 cells. Here we show that Gprc5a, encoding an orphan G protein-coupled receptor, is a novel PTH-inducible gene and negatively regulates osteoblast proliferation and differentiation. PTH treatment induced Gprc5a expression in MC3T3-E1 cells, rat osteosarcoma ROS17/2.8 cells, and mouse femurs. Induction of Gprc5a expression by PTH occurred in the absence of protein synthesis and was mediated primarily via the cAMP pathway, suggesting that Gprc5a is a direct target of PTH signaling. Interestingly, Gprc5a expression was induced additively by co-treatment with PTH and 1α, 25-dihydroxyvitamin D3 (calcitriol), or retinoic acid in MC3T3-E1 cells. Reporter analysis of a 1 kb fragment of human GPRC5A promoter revealed that the promoter fragment showed responsiveness to PTH via the cAMP response element, suggesting that GPRC5A is also a PTH-inducible gene in humans. Gprc5a knockdown promoted cell viability and proliferation, as demonstrated by MTT and BrdU assays. Gprc5a knockdown also promoted osteoblast differentiation, as indicated by gene expression analysis and mineralization assay. Mechanistic studies showed that Gprc5a interacted with BMPR1A and suppressed BMP signaling induced by BMP-2 and constitutively active BMP receptors, ALK2 (ACVR1) Q207D and ALK3 (BMPR1A) Q233D. Thus, our results suggest that Gprc5a is a novel gene induced by PTH that acts in an inhibitory manner on both cell proliferation and osteoblast differentiation and is a candidate for drug targets for osteoporosis.

Role of Piezo1 in modulating the RANKL/OPG ratio in mouse osteoblast cells exposed to Porphyromonas gingivalis lipopolysaccharide and mechanical stress.

Excessive occlusal force with periodontitis leads to rapid alveolar bone resorption. However, the molecular mechanism by which inflammation and mechanical stress cause bone resorption remains unclear. We examined the role of Piezo1, a mechanosensitive ion channel expressed on osteoblasts, in the changes in the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio in mouse MC3T3-E1 osteoblast-like cells under Porphyromonas gingivalis lipopolysaccharide (P.g.-LPS) and mechanical stress. To investigate the effect of P.g.-LPS and mechanical stress on the RANKL/OPG ratio and Piezo1 expression, we stimulated MC3T3-E1 cells with P.g.-LPS. After 3 days in culture, shear stress, a form of mechanical stress, was applied to the cells using an orbital shaker. Subsequently, to investigate the role of Piezo1 in the change of RANKL/OPG ratio, we inhibited Piezo1 function by knockdown via Piezo1 siRNA transfection or by adding GsMTx4, a Piezo1 antagonist. The RANKL/OPG ratio significantly increased in MC3T3-E1 cells cultured in a medium containing P.g.-LPS and undergoing mechanical stress compared to cells treated with P.g.-LPS or mechanical stress alone. However, the expression of Piezo1 was not increased by P.g.-LPS and mechanical stress. In addition, phosphorylation of MEK/ERK was induced in the cells under P.g.-LPS and mechanical stress. MC3T3-E1 cells treated with P.g.-LPS and mechanical stress when cocultured with RAW264.7 cells induced their differentiation into osteoclast-like cells. The increased RANKL/OPG ratio was suppressed by either Piezo1 knockdown or the addition of GsMTx4. Furthermore, GsMTx4 inhibited the phosphorylation of MEK/ERK. These findings suggest that P.g.-LPS and Piezo1-mediated mechanical stress induce MEK/ERK phosphorylation and increase RANKL expression in osteoblasts. Consequently, this leads to the differentiation of osteoclast precursor cells into osteoclasts.

Exposing Differentiated Osteoblast-Like MC3T3-E1 Cells to Extremely Low-Frequency Electric Fields Has Calcification-Promoting Effects

Exposing Differentiated Osteoblast-Like MC3T3-E1 Cells to Extremely Low-Frequency Electric Fields Has Calcification-Promoting Effects

2D magnesium phosphate resorbable coating to enhance cell adhesion on titanium surfaces

Titanium and its alloys are essential metals for orthopedic implant manufacturing due to their exceptional mechanical properties and biocompatibility, used extensively for treating various orthopedic conditions. However, Titanium (Ti) implants have a disadvantage due to lack of bioactivity, potentially affecting osseointegration and osteoconductive capabilities, and may take several months to integrate with bone tissue. In this work, we prepared a layer of 2D magnesium phosphate (MgPi) coating on the surface of titanium surfaces via the spin-coating technique. Various techniques were used to study the phase composition of the coatings, including FTIR, Raman spectroscopy, NMR, and XRD analysis. Morphology and chemical analysis were performed using Atomic force microscopy and SEM/EDX. Nano-scratch test and water contact angle measurements were used to measure adhesion strength and wettability. In addition, in vitro cell assays were used to assess cell adhesion and viability to determine how the MC3T3-E1 osteoblast-like cells reacted to the different treated Ti substrates. AFM results showed that the surface roughness became lower after coatings. MgPi-coated samples showed higher hydrophilicity, protein adsorption, and cell viability than uncoated samples. The nano-scratch test showed that the MgPi coating showed better adherence to chemically and thermally treated samples compared to untreated samples. The deposited MgPi coating has good adhesion to the Ti-substrates. Most significantly, compared to uncoated control (Ti) (p < 0.005) and chemically treated coated samples CT-MgPi (p < 0.005), MC3T3-E1 cell proliferation was significantly increased on thermochemical coated surfaces. These findings point to resorbable two-dimensional MgPi coatings as a potential candidate for promoting Ti implant osseointegration.

Synergistic Effect of Nano Strontium Titanate Coating and Ultraviolet C Photofunctionalization on Osteogenic Performance and Soft Tissue Sealing of poly(ether-ether-ketone).

This study aimed to evaluate the bioactivity of poly(ether ether ketone) (PEEK) after surface modification by persistent photoconductive strontium titanate (SrTiO3) magnetron sputtering and ultraviolet (UV) C irradiation. According to the different modifications, the PEEK specimens were randomly divided into five groups (n = 38/group): PEEK, Sr100-PEEK, Sr200-PEEK, UV/PEEK, and UV/Sr200-PEEK. Then, the specimens of Sr100-PEEK and Sr200-PEEK groups were, respectively, coated with 100 and 200 nm thickness photocatalyst SrTiO3 on the PEEK surface by magnetron sputtering. Subsequently, UV-C light photofunctionalized the specimens of PEEK and Sr200-PEEK groups to form UV/PEEK and UV/Sr200-PEEK groups. The specimens were characterized by a step meter, scanning electron microscopy (SEM), atomic force microscopy (AFM), energy dispersive X-ray spectroscopy (EDX), and a water contact angle meter. The release test of the Sr ion was performed by inductively coupled plasma mass spectrometry (ICP-MS). In vitro study, osteogenic activity (MC3T3-E1 osteoblast-like cells) and epithelial and connective tissue attachment (gingival epithelial cells GE1 and fibroblasts NIH3T3) were analyzed in five groups. Surface morphology of the specimens was changed after coating, and the Sr content on the Sr-PEEK surface was increased with increasing coating thickness. In addition, the contact angle was increased significantly after magnetron sputtering. After UV-C photofunctionalization, the content of surface elements changed and the contact angle was decreased. The release of Sr ion was sustained, and the final cumulative release amount did not exceed the safety limit. In vitro experiments showed that SrTiO3 improved the cell activity of MC3T3-E1 and UV-C irradiation further enhanced the osteogenic performance of PEEK. Besides, UV-C irradiation also significantly promoted the cell viability, development, and expression of adhesion proteins of GE1 and NIH3T3 on PEEK. The present investigation demonstrated that nano SrTiO3 coating with UV-C photofunctionalization synergistically enhanced the osteogenic properties and soft tissue sealing function of PEEK in vitro.

Oncostatin M enhances osteoprotegerin synthesis but reduces macrophage colony‑stimulating factor synthesis in bFGF‑stimulated osteoblast‑like cells.

Bone remodeling is tightly controlled by various factors, including hormones, autacoids and cytokines. Among them, oncostatin M (OSM) is a multifunctional cytokine produced by osteal macrophages, which serves as an essential modulator of bone remodeling. Macrophage colony-stimulating factor (M-CSF) and osteoprotegerin are secreted by osteoblasts, and also have pivotal roles in the regulation of the bone remodeling process. The binding of basic fibroblast growth factor (bFGF), a key regulator of bone remodeling, to the corresponding receptor [fibroblast growth factor receptor (FGFR)] triggers the dimerization and activation of FGFRs, which causes the phosphorylation of FGFR substrates and subsequent activation of downstream effectors, including mitogen-activated protein kinases (MAPKs), via Grb2. bFGF can activate MAPKs, resulting in the synthesis of osteoprotegerin and vascular endothelial growth factor in osteoblast-like MC3T3-E1 cells. In the present study, the effects of OSM on bFGF-induced osteoblast activation were investigated in the synthesis of osteoprotegerin and M-CSF in osteoblasts. The release of osteoprotegerin and M-CSF were analyzed using ELISA. The mRNA expression levels of osteoprotegerin and M-CSF were analyzed using reverse transcription-quantitative PCR. Phosphorylation of p38 MAPK, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 MAPK was assessed using western blotting. OSM enhanced bFGF-induced osteoprotegerin release and bFGF-stimulated mRNA expression of osteoprotegerin. By contrast, OSM suppressed the bFGF-induced release of M-CSF and bFGF-stimulated mRNA expression of M-CSF. SB203580, a p38 MAPK inhibitor, and SP600125, a SAPK/JNK inhibitor, suppressed the bFGF-stimulated M-CSF release, whereas PD98059, an upstream kinase inhibitor of p44/p42 MAPK, failed to suppress the M-CSF release stimulated by bFGF. Furthermore, OSM enhanced the bFGF-induced phosphorylation of p38 MAPK, but attenuated the bFGF-stimulated phosphorylation of SAPK/JNK. By contrast, OSM had little effect on the bFGF-induced phosphorylation of p44/p42 MAPK. SB203580 markedly reduced the amplification of bFGF-stimulated osteoprotegerin release enhanced by OSM. These results strongly suggested that OSM may possess divergent effects on bFGF-induced osteoblast activation, upregulation of p38 MAPK and downregulation of SAPK/JNK, leading to the amplification of osteoprotegerin synthesis and the attenuation of M-CSF synthesis.

Extracellular sodium regulates fibroblast growth factor 23 (FGF23) formation

The bone-derived hormone fibroblast growth factor-23 (FGF23) has recently received much attention due to its association with chronic kidney disease and cardiovascular disease progression. Extracellular sodium concentration ([Na+]) plays a significant role in bone metabolism. Hyponatremia (lower serum [Na+]) has recently been shown to be independently associated with FGF23 levels in patients with chronic systolic heart failure. However, nothing is known about the direct impact of [Na+] on FGF23 production. Here, we show that an elevated [Na+] (+20 mM) suppressed FGF23 formation, whereas low [Na+] (-20 mM) increased FGF23 synthesis in the osteoblast-like cell lines UMR-106 and MC3T3-E1. Similar bidirectional changes in FGF23 abundance were observed when osmolality was altered by mannitol but not by urea, suggesting a role of tonicity in FGF23 formation. Moreover, these changes in FGF23 were inversely proportional to the expression of NFAT5 (nuclear factor of activated T cells-5), a transcription factor responsible for tonicity-mediated cellular adaptations. Furthermore arginine vasopressin (AVP), which is often responsible for hyponatremia, did not affect FGF23 production. Next, we performed a comprehensive and unbiased RNA-seq analysis of UMR-106 cells exposed to low vs. high [Na+], which revealed several novel genes involved in cellular adaptation to altered tonicity. Additional analysis of cells with Crisp-Cas9 mediated NFAT5 deletion indicated that NFAT5 controls numerous genes associated with FGF23 synthesis, thereby confirming its role in [Na+]-mediated FGF23 regulation. In line with these in vitro observations, we found that hyponatremia patients have higher FGF23 levels. Our results suggest that [Na+] is a critical regulator of FGF23 synthesis.

Difference in the Osteoblastic Calcium Signaling Response Between Compression and Stretching Mechanical Stimuli

In orthodontics, various forms of mechanical stimulation induce opposing bone metabolism mechanisms. Bone resorption and bone formation occur in areas of compressive and tensile force action, respectively. The mechanism that causes such a difference in bone metabolism is still unclear. In this study, we investigated the difference in the osteoblastic calcium signaling response between compression and stretching mechanical stimuli. We applied two types of mechanical stimuli to osteoblast-like MC3T3-E1 cells: first microneedle direct indentation onto the cell as compression stimuli, and second stretching stimuli by using originally developed cell stretching MEMS device. Cells were treated with thapsigargin and calcium-free medium to investigate the source of the calcium ion. The results demonstrated variations in the osteoblastic calcium signaling response between the compression and stretching stimuli. The magnitude of an increase in the intracellular calcium ion concentration is much higher in the compression stimuli-applied cell group. Treatment of calcium-free medium nearly suppressed the calcium signaling response to both types of mechanical stimulation. Thapsigargin treatment induced an increase in the magnitude of calcium signaling response to the compression stimuli, while suppressed the slow and sustained increase in the calcium ion concentration in the stretching stimuli-applied cell group. These findings demonstrate the difference in the characteristics of osteoblastic calcium signaling response between compression and stretching mechanical stimuli.

Resveratrol inhibits basic fibroblast growth factor-induced macrophage colony-stimulating factor synthesis via the PI3-kinase/Akt pathway in osteoblasts.

Resveratrol is a natural polyphenol found in grapes and beneficial for human health. Resveratrol regulates basic fibroblast growth factor (bFGF)-induced osteoprotegerin synthesis through Akt pathway in osteoblast-like MC3T3-E1 cells. In this study, we investigated resveratrol effects on bFGF-induced macrophage colony-stimulating factor (M-CSF) synthesis in MC3T3-E1 cells. bFGF significantly stimulated release and mRNA expression of M-CSF, which was reduced by resveratrol and SRT1720, sirtuin 1 (SIRT1) activator. Inauhzin, SIRT1 inhibitor, reversed inhibitory effects of resveratrol on bFGF-induced mRNA expression of M-CSF. Deguelin, Akt inhibitor, and LY294002, phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, reduced bFGF-induced M-CSF synthesis. Inauhzin reversed inhibitory effects of resveratrol on bFGF-induced Akt phosphorylation. Suppressive effect of resveratrol on bFGF-induced osteoprotegerin mRNA expression was confirmed in the identical samples using in experiment of M-CSF mRNA expression. Therefore, resveratrol reduces bFGF-induced M-CSF synthesis in addition to osteoprotegerin synthesis by inhibiting PI3-kinase/Akt pathway and suppressive effects are mediated through SIRT1 activation in osteoblasts.

Oncostatin M stimulates prostaglandin D2-induced osteoprotegerin and interleukin-6 synthesis in osteoblasts

Oncostatin M produced by osteal macrophages plays a significant role in fracture healing. Osteoprotegerin (OPG) secreted by osteoblasts, binds to the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) as a decoy receptor and prevents RANKL from binding to RANK, resulting in bone resorption suppression. Interleukin-6 (IL-6) is a pro-inflammatory cytokine and generally regulates bone resorption. However, accumulating evidence suggests that IL-6 plays pivotal roles in bone formation. We previously showed that prostaglandin D2 (PGD2) induces OPG synthesis by activating p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. Furthermore, we demonstrated that PGD2 stimulates IL-6 synthesis by activating p38 MAP kinase and p44/p42 MAP kinase in MC3T3-E1 cells. In the present study, we investigated whether oncostatin M affects PGD2-stimulated OPG and IL-6 synthesis in MC3T3-E1 cells through MAP kinase activation. The osteoblast-like MC3T3-E1 cells and normal human osteoblasts were treated with oncostatin M and subsequently stimulated with PGD2. Consequently, oncostatin M significantly increased the PGD2-stimulated OPG and IL-6 release in both cells. Oncostatin M significantly enhanced mRNA expression levels of OPG and IL-6 induced by PGD2 similarly in both cells. Regarding the signaling mechanism, oncostatin M did not affect the phosphorylation of p38 MAP kinase, SAPK/JNK, and p44/p42 MAP kinase. Our results suggest that oncostatin M upregulates the PGD2-stimulated OPG and IL-6 synthesis in osteoblasts and therefore affects bone remodeling. However, OPG and IL-6 synthesis are not mediated through p38 MAP kinase, p44/p42 MAP kinase, or SAPK/JNK pathways.

The Influence of Electrode Design on Detecting the Effects of Ferric Ammonium Citrate (FAC) on Pre-Osteoblast through Electrical Cell-Substrate Impedance Sensing (ECIS)

Detection sensitivity is a crucial factor in the application of ECIS sensors. For these biosensors, the electrode configuration has a direct impact on sensitivity, yet few studies on monopolar electrodes have been reported. In this study, ECIS sensor arrays, which have a series of working electrode configuration with a wide diameter range and different electrode number, were fabricated to monitor living osteoblast-like MC3T3-E1 cells. The experimental results revealed that when the electrode diameter was larger than 25 μm, electrodes with smaller diameter and number yielded higher impedance values and generated more impedance shift to cell status change. The membrane capacitance obtained by equivalent circuit fitting was at the same level. When the electrode diameter was even smaller, the results in detection of cell monolayer were opposite, and there was no distinct relationship between impedance and membrane capacitance shift to cell status change and electrode geometry. The proposed sensor chip, allowing for a sustained and stable detection of cellular impedance, provides the basis for the selection of the electrode configuration of monopolar electrodes. The test results of electrodes with a diameter of 25 μm and lower indicated the possibility of single cell impedance measurement, which can provide unique insight into the heterogeneous electrical behavior of cells, and, in this case, the electrode size should be close to the cell size.

Biocompatibility Evaluation of Carbon Nanohorns in Bone Tissues.

With the advent of nanotechnology, the use of nanoparticles as drug delivery system (DDS) has attracted great interest. We aimed to apply carbon nanohorns (CNHs) as DDS in the development of new treatments for bone diseases. We evaluated the in vitro and in vivo cellular responses of CNHs in bone-related cells compared with carbon blacks (CBs), which are similar in particle size but differ in surface and structural morphologies. Although in vitro experiments revealed that both CNHs and CBs were incorporated into the lysosomes of RAW264-induced osteoclast-like cells (OCs) and MC3T3-E1 osteoblast-like cells (OBs), no severe cytotoxicity was observed. CNHs reduced the tartrate-resistant acid phosphatase activity and expression of the differentiation marker genes in OCs at noncytotoxic concentrations, whereas the alkaline phosphatase activity and differentiation of OBs increased. Under calcification of OBs, CNHs increased the number of calcified nodules and were intra- and extracellularly incorporated into calcified vesicles to form crystal nuclei. The in vivo experiments showed significant promotion of bone regeneration in the CNH group alone, with localized CNHs being found in the bone matrix and lacunae. The suppression of OCs and promotion of OBs suggested that CNHs may be effective against bone diseases and could be applied as DDS.

HSP70 inhibitors upregulate prostaglandin E1-induced synthesis of interleukin-6 in osteoblasts.

Interleukin-6 (IL-6) is a pro-inflammatory and bone-resorptive cytokine that also regulates bone formation. We previously showed that prostaglandin E1 (PGE1) induces the synthesis of IL-6 by activating p44/p42 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38 MAPK in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether heat shock protein 70 (HSP70), a molecular chaperone that coordinates protein folding and homeostasis, affects PGE1-stimulated IL-6 synthesis in MC3T3-E1 cells through the MAPK activation. The osteoblast-like MC3T3-E1 cells were treated with HSP70 inhibitors-VER-155008 and YM-08-, PD98059, SB203580 or SP600125 and then stimulated with PGE1. IL-6 synthesis was evaluated using an IL-6 enzyme-linked immunosorbent assay kit. IL-6 mRNA expression was measured by real-time RT-PCR. The phosphorylation of p38 MAPK was evaluated by Western blotting. We found that VER-155008, an HSP70 inhibitor, enhanced the PGE1-stimulated IL-6 release and IL-6 mRNA expression. YM-08, another HSP70 inhibitor, also enhanced PGE1-stimulated IL-6 release. PD98059, a p44/p42 MAPK inhibitor, and SP600125, a SAPK/JNK inhibitor, upregulated PGE1-stimulated IL-6 release. On the other hand, SB203580, a p38 MAPK inhibitor, suppressed PGE1-stimulated IL-6 release. YM-08 stimulated the PGE1-induced phosphorylation of p38 MAPK. SB203580 suppressed the amplification by YM-08 of the PGE1-stimulated IL-6 release. Our results suggest that HSP70 inhibitors upregulate the PGE1-stimulated IL-6 synthesis through p38 MAPK in osteoblasts and therefore affect bone remodeling.

Selective estrogen receptor modulators, acting as agonists of estrogen receptor α in osteoblasts, reduce the TGF-β-induced synthesis of macrophage colony-stimulating factor via inhibition of JNK signaling pathway.

Selective estrogen receptor modulator (SERM) binds to estrogen receptors (ERs) and acts as both an agonist or an antagonist, depending on the target tissue. Raloxifene and bazedoxifene as SERMs are currently used hormone replacement medicines for postmenopausal osteoporosis. Macrophage colony-stimulating factor (M-CSF) secreted from osteoblasts promotes osteoclastogenesis. We have previously demonstrated that transforming growth factor (TGF)-β induces the synthesis of M-CSF via SMAD2/3, p38 mitogen-activated protein kinase (MAPK), p44/p42 MAPK and c-Jun N-terminal kinase (JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether SERM affects the M-CSF synthesis by TGF-β in MC3T3-E1 cells. Raloxifene and bazedoxifene significantly suppressed the synthesis of M-CSF. PPT, an ERα agonist, but not ERB041, an ERβ agonist, inhibited the release of M-CSF. MPP, an ERα antagonist, reversed the suppression by raloxifene of the M-CSF release. Raloxifene attenuated the TGF-β-induced phosphorylation of JNK but not SMAD3, p42 MAPK and p38 MAPK. Bazedoxifene and PPT also inhibited the phosphorylation of JNK. Furthermore, MPP, an ERα antagonist, reversed the suppression by both raloxifene and bazedoxifene of the phosphorylation of JNK. Our results strongly indicate that raloxifene and bazedoxifene, SERMs, suppress the TGF-β-induced synthesis of M-CSF through ERα-mediated inhibition of JNK pathway in osteoblasts.

Low frequency pulsed electromagnetic fields exposure alleviate the abnormal subchondral bone remodeling at the early stage of temporomandibular joint osteoarthritis

BackgroundTemporomandibular joint osteoarthritis (TMJOA) is characterized by abnormal subchondral bone remodeling and cartilage degeneration. As a non-invasive biophysical technology, pulsed electromagnetic field (PEMF) treatment has been proven to be efficient in promoting osteogenesis. However, the potential bone protective effect and mechanism of PEMF on abnormal subchondral bone remodeling in TMJOA are unknown.MethodsUnilateral anterior crossbite (UAC) was used to create TMJOA model in rats, and 17β-estradiol (E2) were injected daily to mimic patients with high-physiological levels of estrogen. Mouse osteoblast-like MC3T3-E1 cells treated with recombinant murine IL-1β was used to establish inflammatory environment in vitro. The treatment group were subjected to PEMF (2.0mT, 15 Hz, 2 h/d). Micro-CT scanning, histological staining, real-time PCR and western blotting assays were preformed to observe the changes in the subchondral bone.ResultsAbnormal resorption of subchondral bone induced by UAC, characterized by decreased bone mineral density, increased osteoclast activity and expression of osteoclast-related factors (RANKL) and down-regulated expression of osteogenesis-related factors (OPG, ALP, Runx2 and OCN) at the early stage, could be reversed by PEMF exposure, which was similar to the effect of estrogen. In addition, PEMF exposure and E2 supplement may have a synergistic effect to some extent. Moreover, PEMF exposure could promote the ALP activity and osteogenic mineralization ability of MC3T3-E1 cells. PEMF promoted the expression of factors related to Wnt/β-Catenin signal pathway both in vivo and in vitro.ConclusionsAppropriate PEMF exposure have a protective effect on subchondral bone in TMJOA at early stage, in which canonical Wnt/β-Catenin pathway may be involved. PEMF may be a promising biophysical approach for early intervention of TMJOA in clinic.

2,3-Diphospho-D-glyceric acid inhibits calciprotein particle growth and calcification in MOVAS cells but not in MC3T3-E1 cells

Anionic polyphosphate compounds are well-known to inhibit the formation of hydroxyapatite crystals. Vascular calcification (VC) is associated with an increase in crystalline calciprotein particles (CPPs) in the bloodstream. 2,3-Diphospho-D-glyceric acid (2,3-DPG) is a highly anionic Poly-P compound found in the concave center of erythrocytes where it binds to hemoglobin, thereby reducing oxygen affinity. Since 2.3-DPG exists in plasma, we hypothesized that 2,3-DPG may act as an endogenous inhibitor of VC. In comparison to widely recognized calcification inhibitors such as myo-inositol hexakisphosphate, 2,3-DPG significantly delayed the formation of crystalline CPPs. Additionally, 2,3-DPG inhibited calcification in a mouse vascular smooth muscle cell line (MOVAS) without cytotoxic effects. Taken together with the result that 2,3-DPG did not impact bone-like nodule formation in mouse osteoblast-like MC3T3-E1 cells, these results suggest that 2,3-DPG may inhibit VC selectively.

Oncostatin M attenuates tumor necrosis factor-α-induced synthesis of macrophage-colony stimulating factor via suppression of Akt in osteoblasts

ABSTRACT Background Oncostatin M produced by osteal macrophages, a cytokine that belongs to the interleukin-6 family, is implicated in bone fracture healing. Macrophage colony-stimulating factor (M-CSF) secreted from osteoblasts plays an important role in osteoclastogenesis. We have previously reported that tumor necrosis factor-α (TNF-α), a potent bone resorptive agent, stimulates the activation of p44/p42 mitogen-activated protein (MAP) kinase, Akt, and p70 S6 kinase in osteoblast-like MC3T3-E1 cells, and induces the synthesis of M-CSF at least in part via Akt. Objective In the present study, we investigated whether oncostatin M affects the TNF-α-induced M-CSF synthesis in MC3T3-E1 cells and the underlying mechanisms. Methods Clonal osteoblast-like MC3T3-E1 cells were treated with oncostatin M or rapamycin and then stimulated with TNF-α. M-CSF release was assessed by ELISA. M-CSF mRNA expression level was assessed by real-time RT-PCR. Phosphorylation of Akt, p44/p42 MAP kinase, and p70 S6 kinase was detected by Western blot analysis. Results Oncostatin M dose-dependently reduced the TNF-α-stimulated M-CSF release. The expression of M-CSF mRNA induced by TNF-α was significantly suppressed by oncostatin M. Rapamycin, an inhibitor of mTOR/p70 S6 kinase, had little effect on the M-CSF release by TNF-α. Oncostatin M significantly reduced the TNF-α-induced phosphorylation of Akt and p44/p42 MAP kinase. However, the p70 S6 kinase phosphorylation by TNF-α was not affected by oncostatin M. Conclusion These results strongly suggest that oncostatin M attenuates TNF-α-stimulated synthesis of M-CSF in osteoblasts, and the inhibitory effect is exerted at a point upstream of Akt and p44/p42 MAP kinase but not p70 S6 kinase.

Oncostatin M reduces the synthesis of macrophage-colony stimulating factor stimulated by TGF-β via suppression of p44/p42 MAP kinase and JNK in osteoblasts

Bone fracture is an important trauma frequently encountered into emergency medicine as well as orthopedics reflecting an aging society. Oncostatin M, an inflammatory cytokine produced by osteal macrophages, has been considered to play a crucial role in fracture healing. Macrophage colony-stimulating factor (M-CSF) secreted from osteoblasts is essential in osteoclastgenesis, and the secretion is stimulated by transforming growth factor-β (TGF-β). The aim of this study is to elucidate the effects of oncostatin M on the TGF-β-induced M-CSF synthesis in osteoblast-like MC3T3-E1 cells and the underlying mechanisms. Oncostatin M attenuated the TGF-β-stimulated M-CSF release and the mRNA expressions. SMAD3 inhibitor SIS3, p38 MAP kinase inhibitor SB203580, MEK1/2 inhibitor PD98059, and SAPK/JNK inhibitor SP600125 significantly suppressed the M-CSF release. Oncostatin M suppressed the TGF-β-induced phosphorylation of p44/p42 MAP kinase and SAPK/JNK, but failed to affect the phosphorylation of SMAD3 and p38 MAP kinase. Oncostatin M attenuated the TGF-β-stimulated vascular endothelial growth factor (VEGF) release and the TGF-β-induced mRNA expressions of VEGF. These results strongly suggest that oncostatin M downregulates TGF-β signaling upstream of p44/p42 MAP kinase and SAPK/JNK, but not SMAD 2/3 and p38 MAP kinase, in osteoblasts, leading to the attenuation of M-CSF synthesis. Our findings might provide a new therapeutic strategy for the acceleration of fracture healing process.