Signaling Pathway In Osteoblasts Research Articles

Osteoblasts are induced into cancer-associated osteoblasts to promote tumor progression in head and neck squamous cell carcinoma

Bone invasion by head and neck squamous cell carcinoma (HNSCC) significantly impacts tumor staging, treatment choice, prognosis, and quality of life. While HNSCC is known to cause osteolytic bone invasion, we found that specific HNSCC subtypes can induce osteogenic bone destruction at the tumor-bone interface. This destruction mode significantly correlated with reduced patient survival rates and increased neck lymph node metastasis. Further in vivo and in vitro experiments indicated that HNSCC cells triggered abnormal phenotypic changes in osteoblasts to remodel the tumor-bone microenvironment, facilitating tumor lymphatic metastasis. Through transcriptome analysis, we identified three genes—osteopontin (SPP1), chemokine (C-X-C motif) ligand 1 (CXCL1), and matrix metalloprotein (MMP)9 (MMP9) linked to a poorer prognosis. We discovered osteoblasts with abnormal phenotypes at the tumor-bone interface exhibiting high SPP1, MMP9, and CXCL1 expressions. Based on these characteristics, we identified this osteoblast subpopulation as “cancer-associated osteoblasts (CAOs).” HNSCC cells activated the TNF-α/NF-κB signaling pathway in osteoblasts, transforming them into “CAOs.” These CAOs significantly contributed to the progression of tumor-induced bone invasion, facilitating cancer growth and metastasis. We first provided clinical data and in vivo and in vitro evidence that HNSCC cells can promote tumor progression by manipulating osteoblasts into “CAOs” in the bone invasion.

SIRT1 Asn346 sugar chain promoting collagen deacetylation protective effect on osteoblasts under stress

Silencing type information regulator homolog 1 (SIRT1) is a class of nicotinamide adenine dinucleotide (NAD+) dependent deacetylases, which is the convergence point of important physiological processes in vivo, namely, osteoblast aging, energy metabolism, and bone remodeling. To verify whether the O-acetylglucosamine (O-GlcNAc) modification of SIRT1 in the nucleus of osteoblasts enhances its deacetylase activity under stress and protects osteoblasts through the RANK/RANKL signaling pathway by collagen deacetylation. The R language and online data research identified SIRT1 as being involved in bone metabolism. Enrichment analysis showed that SIRT1 is involved in osteoblast transcription, apoptosis, and deacetylation pathways. Interactive Immuno-blotting and immunofluorescence experiments revealed that SIRT1 and O-glycosylation catalytic enzyme (OGT) were localized in the nucleus. Mass Spectrometry analysis showed that O-glycosylation occurred on the asparagine at the 346th position of SIRT1, and N346th was located in the central domain of SIRT1. Furthermore, the protein structure analysis of PyMol also proved that the OGT binding region was in the central domain of SIRT1. Under physiological conditions, both wtSIRT1 and SIRT1N346R can inhibit RANKL-mediated transcriptional activation. The RT-PCR detection results showed that wtSIRT1 reduced RANKL transcription under the conditions of apoptotic agent treatment. The finding that SIRT1 can regulate the physiological process of bone remodeling through the RANK/RANKL signaling pathway in osteoblasts under stress. The O-glycosylation and deacetylation activity of SIRT1 significantly increased, regulating the balance between osteoblast survival and apoptosis by deacetylation of key proteins such as RANKL.

Hypoxia-induced Wnt/β-catenin signaling activation in subchondral bone osteoblasts leads to an osteoarthritis-like phenotype of chondrocytes in articular cartilage.

Background: Osteoarthritis (OA) is a whole-joint disease and characterized by alterations in the articular cartilage, subchondral bone, ligaments, and synovial membrane. The crosstalk between cartilage and subchondral bone plays a crucial role in the pathogenesis and progression of OA. Hypoxia has been reported to play an important role in cartilage degradation and subchondral bone remodeling in OA. In this study, we aimed to identify the involvement of hypoxia in modifying the osteoblast phenotypes and determine whether these alterations could influence the metabolism of chondrocytes. Methods: First, the levels of Hif-1α in subchondral bone of different compartments in patients with OA were assessed using immunohistochemistry (IHC). In in vitro, human primary osteoblasts were cultured under hypoxic and normoxic conditions, and the hypoxic or normoxic conditioned media (HCM and NCM) were used to culture human primary chondrocytes. Then, phenotypic changes in osteoblasts were assessed using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Furthermore, the expression of type II collagen (COL2A1), aggrecan (ACAN), SRY-related high-mobility group-box gene 9 (SOX9), matrix metalloproteinase 13 (MMP13), and matrix metalloproteinase 3 (MMP3) in chondrocytes was measured using RT-PCR. Finally, the serum levels of Wnt-related proteins were determined using ELISA. Results: Hif-1α was significantly increased in severely sclerotic subchondral bone compared to less damaged subchondral bone. β-Catenin and SOST were identified as upregulated and downregulated in hypoxic osteoblasts, respectively. The hypoxia-induced results were confirmed by ELISA. Stimulating human primary chondrocytes with HCM significantly induced MMP13 and MMP3 and inhibited COL2A1, ACAN, and SOX9 mRNA expression. The serum levels of DKK-1 were significantly increased in human OA. Conclusion: Together, these findings revealed that hypoxia in subchondral bone is a key factor in the crosstalk between chondrocytes and osteoblasts and facilitates the shift of chondrocytes toward an OA-like phenotype probably by activating the Wnt/β-catenin signaling pathway in osteoblasts.

A GP130-Targeting Small Molecule, LMT-28, Reduces LPS-Induced Bone Resorption around Implants in Diabetic Models by Inhibiting IL-6/GP130/JAK2/STAT3 Signaling.

In this study, we examined the effect of the GP130-targeting molecule, LMT-28, on lipopolysaccharide- (LPS-) induced bone resorption around implants in diabetic models using in vitro and rat animal experiments. First, LMT-28 was added to osteoblasts stimulated by LPS and advanced glycation end products (AGEs), and nuclear factor-κB receptor-activating factor ligand (RANKL) and associated pathways were evaluated. Then, LMT-28 was administered by gavage at 0.23 mg/kg once every 5 days for 2 weeks to type 2 diabetic rats with peri-implantitis induced by LPS injection and silk ligature. The expression of IL-6 and RANKL was evaluated by immunohistochemistry, and the bone resorption around implants was evaluated by microcomputed tomography. The results showed that LMT-28 downregulated the expression of RANKL through the JAK2/STAT3 signaling pathway in osteoblasts stimulated by LPS and AGEs, reduced bone resorption around implants with peri-implantitis, decreased the expression of IL-6 and RANKL, and decreased osteoclast activity in type 2 diabetic rats. This study confirmed the ability of LMT-28 to reduce LPS-induced bone resorption around implants in diabetic rats.

Novel Small Molecule Inhibitor of Osteoclast Differentiation

Osteoporosis results in over a million bone fractures each year despite current therapeutics that inhibit bone resorption. The need for better anti-osteoporotic therapeutics is clear. Recently, extracellular vesicles (EVs) containing RANK that are released by osteoclasts and serve as novel regulators of bone remodeling were identified. These RANK-containing EVs both block bone resorption by osteoclasts and stimulate a RANKL reverse signaling pathway in osteoblasts that promotes bone formation. Small molecules with the same activity could prove to be a new class of therapeutic for treatment of bone disease. To seek such small molecules, a virtual screen identified small molecules that were predicted to bind RANKL in the same location as RANK, and an initial cell culture screen showed eighteen of the candidates reduced osteoclast formation in vitro at a concentration of 100 μM. In this project, one candidate, 3-Nitro-4-phosphobenzoic acid (NPA), was examined in greater detail. NPA does-dependently reduced recombinant RANKL-stimulated differentiation of RAW 264.7 cells into osteoclast-like cells with an IC50 of 38 μM. In calcitriol-stimulated mouse marrow, primary cultures that produce both osteoblasts and osteoclasts, NPA (50 μM) reduced osteoclast formation by 88%, while alkaline phosphatase positive osteoblast numbers were higher. These data show that NPA, in cell culture, is able to simultaneously inhibit osteoclast differentiation and promote osteoblast formation. Thus, NPA is a candidate to be a lead molecule for novel dual-function, small molecule, therapeutic agents to treat osteoporosis.

P9. Sex differences in response to recombinant human bone morphogenetic protein-2 (rhBMP-2) in a rat posterolateral fusion spine model

<h3>BACKGROUND CONTEXT</h3> Estrogen modulates bone activity largely through inhibition of osteoclast bone resorption and bone morphogenetic protein-2 (BMP-2) signaling pathways in osteoblasts. Minimal data exist concerning potential sex-dependent differences in BMP-2-mediated bone regeneration. Here, we aimed to quantify sex-dependent differences in the bone healing response to recombinant human BMP-2 (rhBMP-2) treatment in a rat posterolateral spinal fusion model. <h3>METHODS</h3> A total of 48 female and male Sprague-Dawley rats (N=24/group), underwent L4-L5 posterolateral fusion with bilateral placement of absorbable collagen sponges, loaded with 5 µg of rhBMP-2 (10 µg/animal). At 8 weeks postoperatively, 10 specimens of each sex were tested in flexion-extension with quantification of range of motion (ROM) and stiffness. The remaining specimens were evaluated for new bone growth and fusion via radiography, blinded manual palpation and microcomputed tomography (microCT). Laboratory microCT quantified bone microarchitecture and synchrotron microCT examined bone microstructure. On histology, the number of adipocytes within a standardized field of view was counted for both male and female fusion masses. <h3>RESULTS</h3> Manual palpation scores differed significantly between sexes, with mean fusion scores of 2.4±0.4 in females vs 3.1±0.6 in males, p < 0.001. Biomechanical stiffness did not differ between sexes, but ROM was significantly greater and more variable for females vs males (3.7°±5.6° vs 0.27°±0.15°,p < 0.005, respectively. Laboratory microCT showed significantly smaller volumes of fusion masses in females vs males (262±87 mm³ vs 732±238 mm³, respectively, p < 0.001) but significantly higher bone volume fraction (0.27±0.08 vs 0.12±0.05, respectively, p < 0.001). Mean trabecular thickness was not different, but trabecular number was significantly greater in females (3.1±0.5 mm-1vs 1.5±0.4 mm-1, respectively, p<0.001). Synchrotron microCT showed fine bone structures developing in both sexes at the 8-week time point. Adipocyte density was found to be significantly higher in male fusion masses than in female fusion masses. <h3>CONCLUSIONS</h3> This study demonstrates that sex-based factors may influence both the quantity and quality of bone formation in patients receiving rhBMP-2 for a variety of orthopedic applications. We found that male rats had significantly higher fusion scores, greater fusion-mass volume, and lower ratio of new bone volume to total volume compared to female rats. Male and female mean trabecular thicknesses were equal, but the female fusion masses were more densely filled with trabeculae (higher trabecular number) than those of males. MicroCT results suggested that the male trabeculae were more rod-like and the female more plate-like, although the methods that were used are generally considered less accurate for trabecular bone, which has a high degree of concavity. The incomplete fusions on three female rats, evidenced by high ROM and small gaps on microCT, suggest that females may have a more variable response to rhBMP-2. Investigation into whether these sex-based differences are specific to rhBMP-2-induced bone formation or are more general to bone regeneration/healing is prudent. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Orai1 downregulation causes proliferation reduction and cell cycle arrest via inactivation of the Ras-NF-\u03baB signaling pathway in osteoblasts

BackgroundThe purpose of this study was to determine the role of Orai1 in the regulation of the proliferation and cell cycle of osteoblasts.MethodsThe expression of Orai1 was inhibited by Orai1 small interfering RNA (siRNA) in MC3T3-E1 cells. Following Orai1 downregulation, cell proliferation and cell cycle were examined. Furthermore, the expression of cyclin D1, cyclin E, CDK4, and CDK6 was analyzed. The activity of the Ras-NF-κB signaling pathway was investigated to identify the role of Orai1 in the regulation of osteoblast proliferation.ResultsOrai1 was successfully downregulated in MC3T3-E1 cells by the Orai1 siRNA transfection (p < 0.05). We found that MC3T3-E1 cell proliferation was decreased, and the cell cycle was arrested by Orai1 downregulation (p < 0.05). Additionally, the expression of cyclin D1 was decreased by Orai1 downregulation (p < 0.05), as was the activity of the Ras-NF-κB signaling pathway (p < 0.05). Orai1 siRNA did not further reduce cell proliferation, the proportion of cells in the S phase, and cyclin D1 expression after chemical blockage of the Ras signaling pathway in MC3T3-E1 cells (p > 0.05).ConclusionsThe results reveal that Orai1 downregulation may reduce cyclin D1 expression by inactivating the Ras-NF-κB signaling pathway thus blocking osteoblast proliferation and cell cycle.

The Osteogenesis Imperfecta Type V Mutant BRIL/IFITM5 Promotes Transcriptional Activation of MEF2, NFATc, and NR4A in Osteoblasts.

BRIL (bone restricted ifitm-like; also known as IFITM5) is a transmembrane protein expressed in osteoblasts. Although its role in skeletal development and homeostasis is unknown, mutations in BRIL result in rare dominant forms of osteogenesis imperfecta. The pathogenic mechanism has been proposed to be a gain-of or neomorphic function. To understand the function of BRIL and its OI type V mutant (MALEP BRIL) and whether they could activate signaling pathways in osteoblasts, we performed a luciferase reporter assay screen based on the activity of 26 transcription factors. When overexpressed in MC3T3-E1 and MLO-A5 cells, the MALEP BRIL activated the reporters dependent on MEF2, NFATc, and NR4A significantly more. Additional co-transfection experiments with MEF2C and NFATc1 and a number of their modulators (HDAC4, calcineurin, RCAN, FK506) confirmed the additive or synergistic activation of the pathways by MALEP, and suggested a coordinated regulation involving calcineurin. Endogenous levels of Nr4a members, as well as Ptgs2, were upregulated by MALEP BRIL. Y2H and co-immunoprecipitation indicated that BRIL interacted with CAML, but its contribution as the most upstream stimulator of the Ca2+-calcineurin-MEF2/NFATc cascade was not confirmed convincingly. Altogether the data presented provide the first ever readout to monitor for BRIL activity and suggest a potential gain-of-function causative effect for MALEP BRIL in OI type V, leading to perturbed signaling events and gene expression.

Competitive blocking of LRP4-sclerostin binding interface strongly promotes bone anabolic functions.

Induction of bone formation by Wnt ligands is inhibited when sclerostin (Scl), an osteocyte-produced antagonist, binds to its receptors, the low-density lipoprotein receptor-related proteins 5 or 6 (LRP5/6). Recently, it was shown that enhanced inhibition is achieved by Scl binding to the co-receptor LRP4. However, it is not clear if the binding of Scl to LRP4 facilitates Scl binding to LRP5/6 or inhibits the Wnt pathway in an LRP5/6-independent manner. Here, using the yeast display system, we demonstrate that Scl exhibits a stronger binding affinity for LRP4 than for LRP6. Moreover, we found stronger Scl binding to LRP6 in the presence of LRP4. We further show that a Scl mutant (SclN93A), which tightly binds LRP4 but not LRP6, does not inhibit the Wnt pathway on its own. We demonstrate that SclN93A competes with Scl for a common binding site on LRP4 and antagonizes Scl inhibition of the Wnt signaling pathway in osteoblasts in vitro. Finally, we demonstrate that 2 weeks of bi-weekly subcutaneous injections of SclN93A fused to the fragment crystallizable (Fc) domain of immunoglobulin (SclN93AFc), which retains the antagonistic activity of the mutant, significantly increases bone formation rate and enhances trabecular volumetric bone fraction, trabecular number, and bone length in developing mice. Our data show that LRP4 serves as an anchor that facilitates Scl-LRP6 binding and that inhibition of the Wnt pathway by Scl depends on its prior binding to LRP4. We further provide evidence that compounds that inhibit Scl-LRP4 interactions offer a potential strategy to promote anabolic bone functions.

The Interaction Between Intracellular Energy Metabolism and Signaling Pathways During Osteogenesis.

Osteoblasts primarily mediate bone formation, maintain bone structure, and regulate bone mineralization, which plays an important role in bone remodeling. In the past decades, the roles of cytokines, signaling proteins, and transcription factors in osteoblasts have been widely studied. However, whether the energy metabolism of cells can be regulated by these factors to affect the differentiation and functioning of osteoblasts has not been explored in depth. In addition, the signaling and energy metabolism pathways are not independent but closely connected. Although energy metabolism is mediated by signaling pathways, some intermediates of energy metabolism can participate in protein post-translational modification. The content of intermediates, such as acetyl coenzyme A (acetyl CoA) and uridine diphosphate N-acetylglucosamine (UDP-N-acetylglucosamine), determines the degree of acetylation and glycosylation in terms of the availability of energy-producing substrates. The utilization of intracellular metabolic resources and cell survival, proliferation, and differentiation are all related to the integration of metabolic and signaling pathways. In this paper, the interaction between the energy metabolism pathway and osteogenic signaling pathway in osteoblasts and bone marrow mesenchymal stem cells (BMSCs) will be discussed.

Duloxetine suppresses BMP-4-induced release of osteoprotegerin via inhibition of the SMAD signaling pathway in osteoblasts.

Duloxetine, a selective serotonin-norepinephrine reuptake inhibitor, is currently recommended for the treatment of chronic painful disorders such as fibromyalgia, chronic musculoskeletal pain, and diabetic peripheral neuropathy. We previously demonstrated that bone morphogenetic protein-4 (BMP-4) stimulates osteoprotegerin (OPG) production in osteoblast-like MC3T3-E1 cells, and that p70 S6 kinase positively regulates OPG synthesis. The present study aimed to investigate the effect of duloxetine on BMP-4-stimulated OPG synthesis in these cells. Duloxetine dose-dependently suppressed OPG release stimulated by BMP-4. Fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), reduced BMP-4-stimulated OPG release, whereas a selective and specific norepinephrine reuptake inhibitor, reboxetine, failed to affect OPG release. In addition, another SSRI sertraline also inhibited BMP-4-stimulated OPG release. On the other hand, siRNA of SMAD1 reduced the OPG release stimulated by BMP-4, indicating the involvement of the SMAD1/5/8 pathway in OPG release. Rapamycin inhibited BMP-4-stimulated p70 S6 kinase phosphorylation, and compound C suppressed the SMAD1/5/8 phosphorylation stimulated by BMP-4. Duloxetine did not affect BMP-4-induced phosphorylation of p70 S6 kinase but suppressed SMAD1/5/8 phosphorylation. Both fluvoxamine and sertraline also inhibited BMP-4-elicited phosphorylation of SMAD1/5/8. These results strongly suggest that duloxetine suppresses BMP-4-stimulated OPG release via inhibition of the Smad1/5/8 signaling pathway in osteoblasts.

Promotion of osteointegration under diabetic conditions by a silk fibroin coating on 3D-printed porous titanium implants via a ROS-mediated NF-κB pathway

Clinical evidence indicates the compromised application of titanium implants (TIs) in diabetics, associated with reactive oxygen species (ROS) overproduction at the bone-implant interface. Silk fibroin (SF) has displayed impressive biocompatibility in the application of biomedical material and optimal anti-diabetic effects in oriental medicine. We proposed that SF-coated titanium implants (STIs) could alleviate diabetes-induced compromised osteointegration, which has rarely been reported before. To confirm this hypothesis and explore the underlying mechanisms, rat osteoblasts cultured on 3-dimensional (3D) -printed titanium implants (TIs) and STIs were subjected to normal serum (NS), diabetic serum (DS), DS with N-acetyl-L-cysteine (a ROS inhibitor) or SN50 (an NF-κB inhibitor). An in vivo study was performed on diabetic sheep with TIs or STIs implanted into bone defects on the crista iliaca. The results demonstrated that ROS overproduction induced by diabetes lead to osteoblast dysfunctions and cellular apoptosis on the TI substrate, associated with the activation of an NF-κB signaling pathway in osteoblasts. Importantly, the STI substrate significantly attenuated ROS production and NF-κBp65 phosphorylation, thereby ameliorating the osteoblast biological dysfunctions. These results were further confirmed in vivo by the improved osteointegration of the STIs, as evidenced by Micro-CT and histological examinations compared with those of TIs. These results demonstrated that the ROS-mediated NF-κB signaling pathway played a crucial role in diabetes-induced implant destabilization. Importantly, the SF coating, as a promising material for biomaterial-engineering, markedly improved the clinical treatment effect of TIs under diabetic conditions, possibly associated with the suppression of the NF-κB pathway.

RANKL and RANK in extracellular vesicles: surprising new players in bone remodeling.

Receptor activator of nuclear factor kappa B-ligand (RANKL), its receptor RANK, and osteoprotegerin which binds RANKL and acts as a soluble decoy receptor, are essential controllers of bone remodeling. They also play important roles in establishing immune tolerance and in the development of the lymphatic system and mammary glands. In bone, RANKL stimulates osteoclast formation by binding RANK on osteoclast precursors and osteoclasts. This is required for bone resorption. Recently, RANKL and RANK have been shown to be functional components of extracellular vesicles (EVs). Data linking RANKL and RANK in EVs to biological regulatory roles are reviewed, and crucial unanswered questions are examined. RANKL and RANK are transmembrane proteins and their presence in EVs allows them to act at a distance from their cell of origin. Because RANKL-bearing osteocytes and osteoblasts are often spatially distant from RANK-containing osteoclasts in vivo, this may be crucial for the stimulation of osteoclast formation and bone resorption. RANK in EVs from osteoclasts has the capacity to stimulate a RANKL reverse signaling pathway in osteoblasts that promotes bone formation. This serves to couple bone resorption with bone formation and has inspired novel bifunctional therapeutic agents. RANKL- and RANK- containing EVs in serum may serve as biomarkers for bone and immune pathologies. In summary, EVs containing RANKL and RANK have been identified as intercellular regulators in bone biology. They add complexity to the central signaling network responsible for maintaining bone. RANKL- and RANK-containing EVs are attractive as drug targets and as biomarkers.

Bone morphogenetic protein 9 (BMP9) directly induces Notch effector molecule Hes1 through the SMAD signaling pathway in osteoblasts.

Bone morphogenetic protein (BMP) 9 is one of the most osteogenic BMPs, but its mechanism of action has not been fully elucidated. Hes1, a transcriptional regulator with a basic helix-loop-helix domain, is a well-known effector of Notch signaling. Here, we find that BMP9 induces periodic increases of Hes1 mRNA and protein expression in osteoblasts, presumably through an autocrine negative feedback mechanism. BMP9-mediated Hes1 induction is significantly inhibited by an ALK inhibitor and overexpression of Smad7, an inhibitory Smad. Luciferase and ChIP assays revealed that two Smad-binding sites in the 5' upstream region of the mouse Hes1 gene are essential for transcriptional activation by BMP9. Thus, our data indicate that BMP9 induces Hes1 expression in osteoblasts via the Smad signaling pathway.

CXCL2 attenuates osteoblast differentiation by inhibiting the ERK1/2 signaling pathway.

The C-X-C motif chemokine ligand 2 (CXCL2), a member of the CXC receptor ligand family, is involved in various immune and inflammatory processes, but its effect(s) on bone formation have not yet been reported. We report here that CXCL2 is enriched in bone marrow and show abundant expression of CXCL2 in osteoblasts of osteoporotic mice. CXCL2 neutralization within the bone marrow by using antibody alleviated bone loss in mice, indicating a negative role of CXCL2 in bone formation. In line with this, CXCL2 overexpression attenuated proliferation, as well as differentiation, of osteoblasts in vitro By contrast, CXCL2 downregulation promoted osteoblast expansion and differentiation. Mechanistically, CXCL2 inhibits the ERK1/2 (MAPK3/1) signaling pathway in osteoblasts. Activation of ERK1/2 abolishes the inhibitory effect of CXCL2 in osteoblasts, whereas inactivation of ERK1/2 reverses the osteogenic role of CXCL2 inhibition. These results show that CXCL2 attenuates osteoblast differentiation through inhibition of the ERK1/2 signaling pathway. We demonstrate here that CXCL2 is a negative regulator of bone formation and clarify the responsible mechanisms. Therefore, pharmaceutical coordination of CXCL2 and of the pathways through which it is regulated in osteoblasts might be beneficial regarding bone formation.

Interleukin-35 stimulates tumor necrosis factor-α activated osteoblasts differentiation through Wnt/β-catenin signaling pathway in rheumatoid arthritis

Interleukin (IL)-35 plays an important role in the pathogenesis of rheumatoid arthritis (RA), which is characterized by tumor necrosis factor (TNF)-α activated bone loss beginning early and persisting over time. The aim of this study was to explore the effects and signaling pathway of IL-35 on osteoblasts differentiation in MC3T3E1 cells and TNF-α activated MC3T3E1 cells. A microenvironment was established with low concentration and short-term treatment of TNF-α to mimic inflammatory activated osteoblasts of RA in vitro. The role of IL-35 on osteoblasts proliferation and apoptosis were assessed using cell counting kit (CCK)-8 assay and flow cytometry, respectively. Alkaline phosphatase (ALP) activity was measured by p-nitrophenyl phosphate assay. Extracellular matrix mineralization was measured by Alizarin red S staining. Osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in response to IL-35 were investigated using real-time polymerase chain reaction and western blot analysis. Wnt/β-catenin signaling pathway in osteoblasts was investigated. In basal and TNF-α activated osteoblasts, IL-35 promoted proliferation and inhibited apoptosis. Basal and TNF-α activated ALP activity and mineralization in vitro was increased stimulated by IL-35. Furthermore, IL-35 increased the basal and TNF-α activated OPG expression and decreased basal and TNF-α activated RANKL expression. Blocking Wnt/β-catenin signaling pathway with Dickkopf (Dkk)-1 inhibited the osteogenic effects of IL-35. IL-35 stimulates basal and TNF-α activated osteoblasts differentiation through the Wnt/β-catenin signaling pathway, thus highlighting the IL-35 for pharmaceutical and medicinal applications for treating RA bone loss.

LncRNA UCA1 affects osteoblast proliferation and differentiation by regulating BMP-2 expression.

The aim of this study was to detect the expression of long non-coding ribonucleic acid (lncRNA) urothelial carcinoma associated 1 (UCA1) in the plasma of patients with osteoporosis (OST), and to investigate its influences on the proliferation and differentiation of osteoblasts and its mechanism. Plasma samples were collected from 52 OST patients treated in our hospital and 30 healthy subjects receiving a physical examination, respectively. The expression level of lncRNA UCA1 in OST patients and healthy subjects were detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Furthermore, osteoblast MC3T3-E1 cell lines with a stable knockout of UCA1 in mice were constructed using small-interfering RNA (siRNA). The influence of UCA1 knockout on the proliferation of osteoblasts was detected using cell counting kit-8 (CCK-8) assay. Meanwhile, the proportion of EdU-positive cells in osteoblasts of the control group and UCA1 knockout group was detected using EdU staining. Moreover, the messenger RNA (mRNA) levels of differentiation-related genes, including Runt-related transcription factor 2 (Runx2), Collagen1α1, osteoclast (OC), osteoprotegerin (OPG), osteopontin (OPN) and Osterix (OSX), were detected via RT-PCR. The protein expression level of Runx2 was detected via Western blotting. In addition, osteoblasts were cultured with a bone-derived medium for 14 d. Then, the differentiation status was detected via alizarin red staining and alkaline phosphatase staining. Finally, the expression of bone morphogenetic protein-2 (BMP-2)/(Smad1/5/8) signaling pathway was analyzed using Western blotting. The expression of plasma lncRNA UCA1 was significantly increased in OST patients (p<0.05). Cell experiments revealed that UCA1 siRNA intervention could significantly promote the proliferation and differentiation of osteoblast MC3T3-E1 cell lines. In addition, Western blotting showed that the pro-apoptotic effect of UCA1 might be mediated by the BMP-2/(Smad1/5/8) signaling pathway in osteoblasts. Inhibiting lncRNA UCA1 can promote the proliferation and differentiation of osteoblasts by activating the BMP-2/(Smad1/5/8) signaling pathway in osteoblasts. Therefore, UCA1 is expected to be a new therapeutic target for OST.

Effects of lncRNA DANCR on proliferation and differentiation of osteoblasts by regulating the Wnt/β-catenin pathway.

To measure the expression level of long non-coding ribonucleic acids (lncRNAs) differentiation antagonizing non-protein coding RNA (DANCR) in serum of patients with fracture and investigate its influences on the proliferation and differentiation of osteoblasts. Serum samples were collected from 44 fracture patients treated in our hospital and 24 healthy people receiving physical examination in our hospital. Then, reverse transcription-polymerase chain reaction (RT-PCR) technique was used to detect the expression of lncRNA DANCR in the serum of patients with fracture and healthy subjects. MC3T3-E1 mouse osteoblast cell line with stably-knocked out DANCR was further established using small interfering RNAs (siRNAs), and the effect of DANCR knockout on the proliferation of osteoblasts was determined using cell counting kit-8 (CCK-8). At the same time, 5-Ethynyl-2'-deoxyuridine (EdU) staining assay was performed to detect the percentage of EdU-positive cells in osteoblasts in control group and DANCR knockout group. In addition, the mRNA levels of differentiation-related genes including Runt-related transcription factor 2 (Runx2), Collagen1α1, osteocalcin (OC) and osterix (OSX) were detected via RT-PCR, and the protein level of Runx2 was measured through Western blotting. Moreover, osteoblasts were cultured with osteogenic medium for 14 d, and then alizarin red staining and alkaline phosphatase (ALP) staining assays were carried out to examine the differentiation of these osteoblasts. Lastly, Western blotting technique was employed to analyze the expression of the Wnt/β-catenin signaling pathway. The expression of lncRNA DANCR was significantly increased in the serum of fracture patients (p<0.05). The results of in-vitro cell experiments showed that the intervention of DANCR with siRNA was able to clearly promote the proliferation and differentiation of MC3T3-E1 osteoblast cell line. According to the results of Western blotting, DANCR promoted the apoptosis and proliferation, which was mediated by the activated Wnt/β-catenin signaling pathway in osteoblasts. LncRNA DANCR inhibition can facilitate the proliferation and differentiation of osteoblasts by activating the Wnt/β-catenin signaling pathway in osteoblasts. Therefore, DANCR is expected to be a new target promoting fracture healing.

Correction to Antiosteoporotic Activity of Harpagoside by Upregulation of the BMP2 and Wnt Signaling Pathways in Osteoblasts and Suppression of Differentiation in Osteoclasts.

Correction to Antiosteoporotic Activity of Harpagoside by Upregulation of the BMP2 and Wnt Signaling Pathways in Osteoblasts and Suppression of Differentiation in Osteoclasts.

Porcine Bone Incorporated With 4-Hexylresorcinol Increases New Bone Formation by Suppression of the Nuclear Factor Kappa B Signaling Pathway.

The objectives of this study were to evaluate the suppression of the nuclear factor kappa B (NF-kB) pathway by 4-hexylresorcinol (4HR), which was activated by tumor necrosis factor-α (TNF-α) in osteoblasts, and new bone formation by 4HR-incorporated porcine bone in an animal model. For the confirmation of successful incorporation of 4HR into porcine bone, scanning electron microscopy (SEM) and Fourier transform-infrared (FT-IR) analysis were performed. High performance liquid chromatography was performed for the analysis of the 4HR release profile from porcine bone. MC 3T3-E1 cells were used for the analysis of the NF-kB signaling pathway activation by western blotting and real-time reverse transcriptase polymerase chain reaction. New bone formation and the analysis of marker protein expression were studied in a rat calvarial critical-sized defect model. Both SEM and FT-IR analysis demonstrated successful incorporation of 4HR into porcine bone. Approximately 30% of 4HR was steadily released from porcine bone for 18 days. 4HR suppressed the NF-kB signaling pathway, which was activated by TNF-α application in MC 3T3-E1 cells. Histological analysis revealed that porcine bone particles with incorporated 4HR showed significantly greater new bone formation than those without 4HR at 4 and 8 weeks after operation (P < 0.05). The expression intensities of alkaline phosphatase, osteoprotegerin, and osteocalcin were also higher in the 4HR-incorporated group. The application of 4HR suppressed the NF-kB signaling pathway in osteoblasts and 4HR-containing porcine bone particles promoted new bone formation in a rat calvarial defect model.