TMARg, a Novel Anthraquinone Isolated from Rubia cordifolia Nakai, Increases Osteogenesis and Mineralization through BMP2 and β-Catenin Signaling

Background: Plant extracts have long been regarded as useful medicines in the treatment of human diseases. Rubia cordifolia Nakai has been used as a traditional medicine, as it has pharmacological properties such as antioxidant and anti-inflammatory activity. However, the biological functions of TMARg, isolated from the roots of R. cordifolia, in osteoblast differentiation remain unknown. This study was performed to investigate the pharmacological effects and intracellular signaling of TMARg in the osteoblast differentiation of pre-osteoblast MC3T3-E1 cells and mesenchymal precursor C2C12 cells. Methods: Cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP), and by staining it with Alizarin red S (ARS). Cell migration was determined by using migration assays. Western blot analysis and immunocytochemical analysis were used to examine the intracellular signaling pathways and differentiation proteins. Results: In the present study, TMARg showed no cytotoxicity and increased the osteoblast differentiation in pre-osteoblasts, as assessed from the alkaline phosphate (ALP) staining and activity and ARS staining. TMARg also induced BMP2 expression and increased the p-smad1/5/8-RUNX2 and β-catenin pathways in both MC3T3-E1 and C2C12 cells. Furthermore, TMARg activated mitogen-activated protein kinases (MAPKs) and increased the cell migration rate. In addition, the TMARg-mediated osteoblast differentiation was suppressed by BMP and Wnt inhibitors with the downregulation of BMP2 expression. Conclusion: These findings demonstrate that TMARg exerts pharmacological and biological effects on osteoblast differentiation through the activation of BMP2 and β-catenin signaling pathways, and suggest that TMARg might be a potential phytomedicine for the treatment of bone diseases.

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7-HYB, a Phenolic Compound Isolated from Myristica fragrans Houtt Increases Cell Migration, Osteoblast Differentiation, and Mineralization through BMP2 and β-catenin Signaling

International Journal of Molecular Sciences ◽

10.3390/ijms21218059 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8059

Author(s):

Kyung-Ran Park ◽

Yoon-Ju Kwon ◽

Ji-Eun Park ◽

Hyung-Mun Yun

Keyword(s):

Cell Migration ◽

Phenolic Compound ◽

Osteoblast Differentiation ◽

Bone Mineralization ◽

Biological Effects ◽

Bone Diseases ◽

Myristica Fragrans ◽

Receptor Activator ◽

Mitogen Activated Protein ◽

Myristica Fragrans Houtt

The seeds (nutmegs) of Myristica fragrans Houtt have been used as popular spices and traditional medicine to treat a variety of diseases. A phenolic compound, ((7S)-8′-(benzo[3′,4′]dioxol-1′-yl)-7-hydroxypropyl)benzene-2,4-diol (7-HYB) was isolated from the seeds of M. fragrans. This study aimed to investigate the anabolic effects of 7-HYB in osteogenesis and bone mineralization. In the present study, 7-HYB promotes the early and late differentiation of MC3T3-E1 preosteoblasts. 7-HYB also elevated cell migration rate during differentiation of the preosteoblasts with the increased phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK1/2, p38, and JNK. In addition, 7-HYB induced the protein level of BMP2, the phosphorylation of Smad1/5/8, and the expression of RUNX2. 7-HYB also inhibited GSK3β and subsequently increased the level of β-catenin. However, in bone marrow macrophages (BMMs), 7-HYB has no biological effects in cell viability, TRAP-positive multinuclear osteoclasts, and gene expression (c-Fos and NF-ATc1) in receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. Our findings suggest that 7-HYB plays an important role in osteoblast differentiation through the BMP2 and β-catenin signaling pathway. It also indicates that 7-HYB might have a therapeutic effect for the treatment of bone diseases such as osteoporosis and periodontitis.

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Ziyuglycoside I Upregulates RUNX2 through ERK1/2 in Promoting Osteoblast Differentiation and Bone Mineralization

The American Journal of Chinese Medicine ◽

10.1142/s0192415x21500427 ◽

2021 ◽

pp. 1-18

Author(s):

Kyung-Ran Park ◽

Joon Yeop Lee ◽

MyoungLae Cho ◽

Hyung-Mun Yun

Keyword(s):

Cell Migration ◽

Osteoblast Differentiation ◽

Bone Mineralization ◽

Biological Effects ◽

Bone Diseases ◽

Nuclear Accumulation ◽

Herbaceous Plant ◽

Mrna Levels ◽

Dependent Manner ◽

Alizarin Red

Sanguisorba officinalis L. (Rosaceae) is a perennial herbaceous plant and its roots have been used as an important traditional medicine for over 2000 years. Ziyuglycoside I (Ziyu), an active compound isolated from the roots of S. officinalis L., has shown biological effects such as anti-oxidant, antiviral, and antiwrinkle activities. This study aimed to elucidate the underlying mechanisms of action of Ziyu on cytotoxicity, migration, and differentiation of pre-osteoblasts. Herein, at concentrations ranging from 1 to 100 [Formula: see text]M, Ziyu was not cytotoxic against pre-osteoblasts. Alkaline phosphatase activity assay and staining, and migration assay showed that Ziyu increased cell migration and promoted early osteoblast differentiation, followed by the enhancement of mineralized nodule formation in a dose-dependent manner, as indicated by Alizarin Red S staining. In addition, Ziyu increased the protein levels of runt-related transcription factor 2 (RUNX2) during osteoblast differentiation, whereas it did not affect the phosphorylation of Smad1/5/8 and GSK3b and expression of [Formula: see text]-catenin. Ziyu also activated ERK1/2 and mitogen-activated protein kinase during osteoblast differentiation, and ERK1/2 inhibitor attenuated Ziyu-mediated RUNX2 expression and nuclear accumulation. Furthermore, Ziyu-mediated early and late osteoblast differentiation was significantly suppressed by the inhibition of ERK1/2, which was accompanied by attenuation in the mRNA levels of osteoblast-related genes including bone sialoprotein, osteopontin, and osteocalcin. Taken together, the findings of this study provide evidence that Ziyu promotes cell migration, osteoblast differentiation, and bone mineralization and suggest a potential role for Ziyu in the treatment of bone diseases.

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Osteogenic Effects of Triterpene Saponin Soyasapogenol B on Differentiation, Mineralization, Autophagy, and Necroptosis

10.21203/rs.3.rs-1182818/v1 ◽

2021 ◽

Author(s):

Kyung-Ran Park ◽

Joon Yeop Lee ◽

Soo Hyun Kim ◽

Il Keun Kwon ◽

Hyung-Mun Yun

Keyword(s):

Cell Proliferation ◽

Osteoblast Differentiation ◽

Biological Effects ◽

Bone Diseases ◽

Adhesion Assay ◽

Pharmacological Properties ◽

Alizarin Red ◽

Triterpenoid Saponins ◽

Triterpene Saponin ◽

Osteogenic Activity

Abstract Background: Triterpenoid saponins are a diverse group of natural compounds in plants. A triterpene saponin, Soyasapogenol B (SoyB), from Arachis hypogaea (peanut) has various pharmacological properties. This study aimed to elucidate pharmacological properties and mechanisms of SoyB on bone-forming cells. Methods: Cell viability adhesion, and migration were analyzed using MTT assay, cell adhesion assay, and Boyden chamber assay. Osteogenic activity and osteogenicity were analyzed using alkaline phosphatase (ALP) staining and activity, and Alizarin Red S (ARS) staining. Cell signaling, protein expression, and autophagy were analyzed using Western blot analysis, immunofluorescence assay, and DAPGreen autophagy detection assay. Results and Conclusion: In the present study, SoyB (> 99.99% purity), triterpene saponin, was isolated from the fruit of A. hypogaea. At concentrations ranging from 1 to 20 mM, SoyB showed no cell proliferation effects, whereas 30 - 100 mM SoyB increased cell proliferation in MC3T3-E1 cells. Next, osteoblast differentiation was analyzed and found that SoyB enhanced ALP staining and activity and bone mineralization as evidence for early and late osteoblast differentiation. SoyB also induced RUNX2 expression in nucleus with the increased phosphorylation of Smad1/5/8 and JNK2 during osteoblast differentiation. In addition, SoyB-mediated osteoblast differentiation was not associated with autophagy and necroptosis. Furthermore, SoyB increased cell migration and adhesion with the upregulation of MMP13 levels during osteoblast differentiation. The findings of this study provide new evidence that SoyB possesses biological effects on osteogenic activity and osteogenicity in bone-forming cells, and suggest a potentially beneficial role for peanuts foods and drugs containing SoyB in the treatment and prevention of bone diseases.

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Myostatin Deficiency Protects C2C12 Cells from Oxidative Stress by Inhibiting Intrinsic Activation of Apoptosis

Cells ◽

10.3390/cells10071680 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1680

Author(s):

Marius Drysch ◽

Sonja Verena Schmidt ◽

Mustafa Becerikli ◽

Felix Reinkemeier ◽

Stephanie Dittfeld ◽

...

Keyword(s):

Intracellular Signaling ◽

Ischemia Reperfusion ◽

C2c12 Cell ◽

C2c12 Cells ◽

Ros Generation ◽

Protective Effects ◽

Activity Increase ◽

Mitogen Activated Protein ◽

Myostatin Inhibition ◽

Hypoxia Reoxygenation

Ischemia reperfusion (IR) injury remains an important topic in clinical medicine. While a multitude of prophylactic and therapeutic strategies have been proposed, recent studies have illuminated protective effects of myostatin inhibition. This study aims to elaborate on the intracellular pathways involved in myostatin signaling and to explore key proteins that convey protective effects in IR injury. We used CRISPR/Cas9 gene editing to introduce a Myostatin (Mstn) deletion into a C2C12 cell line. In subsequent experiments, we evaluated overall cell death, activation of apoptotic pathways, ROS generation, lipid peroxidation, intracellular signaling via mitogen-activated protein kinases (MAPKs), cell migration, and cell proliferation under hypoxic conditions followed by reoxygenation to simulate an IR situation in vitro (hypoxia reoxygenation). It was found that mitogen-activated protein kinase kinase 3/6, also known as MAPK/ERK Kinase 3/6 (MEK3/6), and subsequent p38 MAPK activation were blunted in C2C12-Mstn−/− cells in response to hypoxia reoxygenation (HR). Similarly, c-Jun N-terminal kinase (JNK) activation was negated. We also found the intrinsic activation of apoptosis to be more important in comparison with the extrinsic activation. Additionally, intercepting myostatin signaling mitigated apoptosis activation. Ultimately, this research validated protective effects of myostatin inhibition in HR and identified potential mediators worth further investigation. Intercepting myostatin signaling did not inhibit ROS generation overall but mitigated cellular injury. In particular, intrinsic activation of apoptosis origination from mitochondria was alleviated. This was presumably mediated by decreased activation of p38 caused by the diminished kinase activity increase of MEK3/6. Overall, this work provides important insights into HR signaling in C2C12-Mstn−/− cells and could serve as basis for further research.

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TRAF6-Dependent Mitogen-Activated Protein Kinase Activation Differentially Regulates the Production of Interleukin-12 by Macrophages in Response to Toxoplasma gondii

Infection and Immunity ◽

10.1128/iai.72.10.5662-5667.2004 ◽

2004 ◽

Vol 72 (10) ◽

pp. 5662-5667 ◽

Cited By ~ 43

Author(s):

Nicola J. Mason ◽

Jim Fiore ◽

Takashi Kobayashi ◽

Katherine S. Masek ◽

Yongwon Choi ◽

...

Keyword(s):

Protein Kinase ◽

Toxoplasma Gondii ◽

Signaling Pathways ◽

Intracellular Signaling ◽

Mitogen Activated Protein Kinase ◽

Interleukin 12 ◽

Wild Type ◽

Kinase Activation ◽

Extracellular Signal ◽

Mitogen Activated Protein

ABSTRACT The production of interleukin-12 (IL-12) is critical to the development of innate and adaptive immune responses required for the control of intracellular pathogens. Many microbial products signal through Toll-like receptors (TLR) and activate NF-κB family members that are required for the production of IL-12. Recent studies suggest that components of the TLR pathway are required for the production of IL-12 in response to the parasite Toxoplasma gondii; however, the production of IL-12 in response to this parasite is independent of NF-κB activation. The adaptor molecule TRAF6 is involved in TLR signaling pathways and associates with serine/threonine kinases involved in the activation of both NF-κB and mitogen-activated protein kinase (MAPK). To elucidate the intracellular signaling pathways involved in the production of IL-12 in response to soluble toxoplasma antigen (STAg), wild-type and TRAF6−/− mice were inoculated with STAg, and the production of IL-12(p40) was determined. TRAF6−/− mice failed to produce IL-12(p40) in response to STAg, and TRAF6−/− macrophages stimulated with STAg also failed to produce IL-12(p40). Studies using Western blot analysis of wild-type and TRAF6−/− macrophages revealed that stimulation with STAg resulted in the rapid TRAF6-dependent phosphorylation of p38 and extracellular signal-related kinase, which differentially regulated the production of IL-12(p40). The studies presented here demonstrate for the first time that the production of IL-12(p40) in response to toxoplasma is dependent upon TRAF6 and p38 MAPK.

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Signaling through mitogen-activated protein kinase and Rac/Rho does not duplicate the effects of activated Ras on skeletal myogenesis.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.3547 ◽

1997 ◽

Vol 17 (7) ◽

pp. 3547-3555 ◽

Cited By ~ 59

Author(s):

M B Ramocki ◽

S E Johnson ◽

M A White ◽

C L Ashendel ◽

S F Konieczny ◽

...

Keyword(s):

Map Kinase ◽

Signaling Pathways ◽

Transcriptional Activation ◽

Intracellular Signaling ◽

Mitogen Activated Protein Kinase ◽

Ras Signaling ◽

Negative Effects ◽

Intracellular Signaling Pathway ◽

Helix Loop Helix ◽

Mitogen Activated Protein

The ability of basic helix-loop-helix muscle regulatory factors (MRFs), such as MyoD, to convert nonmuscle cells to a myogenic lineage is regulated by numerous growth factor and oncoprotein signaling pathways. Previous studies have shown that H-Ras 12V inhibits differentiation to a skeletal muscle lineage by disrupting MRF function via a mechanism that is independent of the dimerization, DNA binding, and inherent transcriptional activation properties of the proteins. To investigate the intracellular signaling pathway(s) that mediates the inhibition of MRF-induced myogenesis by oncogenic Ras, we tested two transformation-defective H-Ras 12V effector domain variants for their ability to alter terminal differentiation. H-Ras 12V,35S retains the ability to activate the Raf/MEK/mitogen-activated protein (MAP) kinase cascade, whereas H-Ras 12V,40C is unable to interact directly with Raf-1 yet still influences other signaling intermediates, including Rac and Rho. Expression of each H-Ras 12V variant in C3H10T1/2 cells abrogates MyoD-induced activation of the complete myogenic program, suggesting that MAP kinase-dependent and -independent Ras signaling pathways individually block myogenesis in this model system. However, additional studies with constitutively activated Rac1 and RhoA proteins revealed no negative effects on MyoD-induced myogenesis. Similarly, treatment of Ras-inhibited myoblasts with the MEK1 inhibitor PD98059 revealed that elevated MAP kinase activity is not a significant contributor to the H-Ras 12V effect. These data suggest that an additional Ras pathway, distinct from the well-characterized MAP kinase and Rac/Rho pathways known to be important for the transforming function of activated Ras, is primarily responsible for the inhibition of myogenesis by H-Ras 12V.

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Effects of PIN on Osteoblast Differentiation and Matrix Mineralization through Runt-Related Transcription Factor

International Journal of Molecular Sciences ◽

10.3390/ijms21249579 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9579

Author(s):

Kyung-Ran Park ◽

SooHyun Kim ◽

MyoungLae Cho ◽

Sang Wook Kang ◽

Hyung-Mun Yun

Keyword(s):

Transcription Factor ◽

Cell Migration ◽

Protein Level ◽

Osteoblast Differentiation ◽

Signaling Molecules ◽

Dependent Manner ◽

Alizarin Red ◽

Matrix Mineralization ◽

Related Transcription Factor ◽

Dose Dependent

Styrax Japonica Sieb. et Zucc. has been used as traditional medicine in inflammatory diseases, and isolated compounds have shown pharmacological activities. Pinoresinol glucoside (PIN) belonging to lignins was isolated from the stem bark of S. Japonica. This study aimed to investigate the biological function and mechanisms of PIN on cell migration, osteoblast differentiation, and matrix mineralization. Herein, we investigated the effects of PIN in MC3T3-E1 pre-osteoblasts, which are widely used for studying osteoblast behavior in in vitro cell systems. At concentrations ranging from 0.1 to 100 μM, PIN had no cell toxicity in pre-osteoblasts. Pre-osteoblasts induced osteoblast differentiation, and the treatment of PIN (10 and 30 μM) promoted the cell migration rate in a dose-dependent manner. At concentrations of 10 and 30 μM, PIN elevated early osteoblast differentiation in a dose-dependent manner, as indicated by increases in alkaline phosphatase (ALP) staining and activity. Subsequently, PIN also increased the formation of mineralized nodules in a dose-dependent manner, as indicated by alizarin red S (ARS) staining, demonstrating positive effects of PIN on late osteoblast differentiation. In addition, PIN induced the mRNA level of BMP2, ALP, and osteocalcin (OCN). PIN also upregulated the protein level of BMP2 and increased canonical BMP2 signaling molecules, the phosphorylation of Smad1/5/8, and the protein level of Runt-related transcription factor 2 (RUNX2). Furthermore, PIN activated non-canonical BMP2 signaling molecules, activated MAP kinases, and increased β-catenin signaling. The findings of this study indicate that PIN has biological roles in osteoblast differentiation and matrix mineralization, and suggest that PIN might have anabolic effects in bone diseases such as osteoporosis and periodontitis.

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Influence of Culture Period on Osteoblast Differentiation of Tissue-Engineered Bone Constructed by Apatite-Fiber Scaffolds Using Radial-Flow Bioreactor

International Journal of Molecular Sciences ◽

10.3390/ijms222313080 ◽

2021 ◽

Vol 22 (23) ◽

pp. 13080

Author(s):

Kitaru Suzuki ◽

Jun Fukasawa ◽

Maiko Miura ◽

Poon Nian Lim ◽

Michiyo Honda ◽

...

Keyword(s):

Osteoblast Differentiation ◽

Radial Flow ◽

Bone Marrow Cells ◽

Three Dimensional ◽

3D Cell Culture ◽

Bone Diseases ◽

Culture Period ◽

Alizarin Red ◽

Fiber Scaffolds ◽

Radial Flow Bioreactor

With the limitation of autografts, the development of alternative treatments for bone diseases to alleviate autograft-related complications is highly demanded. In this study, a tissue-engineered bone was formed by culturing rat bone marrow cells (RBMCs) onto porous apatite-fiber scaffolds (AFSs) with three-dimensional (3D) interconnected pores using a radial-flow bioreactor (RFB). Using the optimized flow rate, the effect of different culturing periods on the development of tissue-engineered bone was investigated. The 3D cell culture using RFB was performed for 0, 1 or 2 weeks in a standard medium followed by 0, 1 or 2 weeks in a differentiation medium. Osteoblast differentiation in the tissue-engineered bone was examined by alkaline phosphatase (ALP) and osteocalcin (OC) assays. Furthermore, the tissue-engineered bone was histologically examined by hematoxylin and eosin and alizarin red S stains. We found that the ALP activity and OC content of calcified cells tended to increase with the culture period, and the differentiation of tissue-engineered bone could be controlled by varying the culture period. In addition, the employment of RFB and AFSs provided a favorable 3D environment for cell growth and differentiation. Overall, these results provide valuable insights into the design of tissue-engineered bone for clinical applications.

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Skeletal (stromal) stem cells: An update on intracellular signaling pathways controlling osteoblast differentiation

Bone ◽

10.1016/j.bone.2014.07.028 ◽

2015 ◽

Vol 70 ◽

pp. 28-36 ◽

Cited By ~ 62

Author(s):

Basem M. Abdallah ◽

Abbas Jafari ◽

Walid Zaher ◽

Weimin Qiu ◽

Moustapha Kassem

Keyword(s):

Stem Cells ◽

Signaling Pathways ◽

Intracellular Signaling ◽

Osteoblast Differentiation ◽

Intracellular Signaling Pathways ◽

Stromal Stem Cells

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Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.4930 ◽

1995 ◽

Vol 15 (9) ◽

pp. 4930-4938 ◽

Cited By ~ 164

Author(s):

R Zinck ◽

M A Cahill ◽

M Kracht ◽

C Sachsenmaier ◽

R A Hipskind ◽

...

Keyword(s):

Transcription Factor ◽

Protein Synthesis ◽

Protein Kinase ◽

Signaling Pathways ◽

Intracellular Signaling ◽

Mitogen Activated Protein Kinase ◽

Early Gene ◽

Immediate Early ◽

Protein Synthesis Inhibitors ◽

Mitogen Activated Protein

Inhibitors of protein synthesis, such as anisomycin and cycloheximide, lead to superinduction of immediate-early genes. We demonstrate that these two drugs activate intracellular signaling pathways involving both the mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) cascades. The activation of either pathway correlates with phosphorylation of the c-fos regulatory transcription factor Elk-1. In HeLa cells, anisomycin stabilizes c-fos mRNA when protein synthesis is inhibited to only 50%. Under these conditions, anisomycin, in contrast to cycloheximide, rapidly induces kinase activation and efficient Elk-1 phosphorylation. However, full inhibition of translation by either drug leads to prolonged activation of SAPK activity, while MAPK induction is transient. This correlates with prolonged Elk-1 phosphorylation and c-fos transcription. Elk-1 induction and c-fos activation are also observed in KB cells, in which anisomycin strongly induces SAPKs but not MAPKs. Purified p54 SAPK alpha efficiently phosphorylates the Elk-1 C-terminal domain in vitro and comigrates with anisomycin-activated kinases in in-gel kinase assays. Thus, Elk-1 provides a potential convergence point for the MAPK and SAPK signaling pathways. The activation of signal cascades and control of transcription factor function therefore represent prominent processes in immediate-early gene superinduction.

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