RUNX2-modifying enzymes: therapeutic targets for bone diseases

Abstract RUNX2 is a master transcription factor of osteoblast differentiation. RUNX2 expression in the bone and osteogenic front of a suture is crucial for cranial suture closure and membranous bone morphogenesis. In this manner, the regulation of RUNX2 is precisely controlled by multiple posttranslational modifications (PTMs) mediated by the stepwise recruitment of multiple enzymes. Genetic defects in RUNX2 itself or in its PTM regulatory pathways result in craniofacial malformations. Haploinsufficiency in RUNX2 causes cleidocranial dysplasia (CCD), which is characterized by open fontanelle and hypoplastic clavicles. In contrast, gain-of-function mutations in FGFRs, which are known upstream stimulating signals of RUNX2 activity, cause craniosynostosis (CS) characterized by premature suture obliteration. The identification of these PTM cascades could suggest suitable drug targets for RUNX2 regulation. In this review, we will focus on the mechanism of RUNX2 regulation mediated by PTMs, such as phosphorylation, prolyl isomerization, acetylation, and ubiquitination, and we will summarize the therapeutics associated with each PTM enzyme for the treatment of congenital cranial suture anomalies.

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Biological Mechanisms of Paeonoside in the Differentiation of Pre-Osteoblasts and the Formation of Mineralized Nodules

International Journal of Molecular Sciences ◽

10.3390/ijms22136899 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6899

Author(s):

Kyung-Ran Park ◽

Joon Yeop Lee ◽

Myounglae Cho ◽

Jin Tae Hong ◽

Hyung-Mun Yun

Keyword(s):

Osteoblast Differentiation ◽

Bone Diseases ◽

Bone Morphogenetic Protein 2 ◽

Nodule Formation ◽

Biological Mechanisms ◽

Cytotoxic Effects ◽

Runx2 Expression ◽

Migration Assay ◽

Morphogenetic Protein ◽

Related Transcription Factor

Paeonia suffruticosa is a magnificent and long-lived woody plant that has traditionally been used to treat various diseases including inflammatory, neurological, cancer, and cardiovascular diseases. In the present study, we demonstrated the biological mechanisms of paeonoside (PASI) isolated from the dried roots of P. suffruticosa in pre-osteoblasts. Herein, we found that PASI has no cytotoxic effects on pre-osteoblasts. Migration assay showed that PASI promoted wound healing and transmigration in osteoblast differentiation. PASI increased early osteoblast differentiation and mineralized nodule formation. In addition, PASI enhanced the expression of Wnt3a and bone morphogenetic protein 2 (BMP2) and activated their downstream molecules, Smad1/5/8 and β-catenin, leading to increases in runt-related transcription factor 2 (RUNX2) expression during osteoblast differentiation. Furthermore, PASI-mediated osteoblast differentiation was attenuated by inhibiting the BMP2 and Wnt3a pathways, which was accompanied by reduction in the expression of RUNX2 in the nucleus. Taken together, our findings provide evidence that PASI enhances osteoblast differentiation and mineralized nodules by regulating RUNX2 expression through the BMP2 and Wnt3a pathways, suggesting a potential role for PASI targeting osteoblasts to treat bone diseases including osteoporosis and periodontitis.

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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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VPTMdb: a viral posttranslational modification database

Briefings in Bioinformatics ◽

10.1093/bib/bbaa251 ◽

2020 ◽

Author(s):

Yujia Xiang ◽

Quan Zou ◽

Lilin Zhao

Keyword(s):

Posttranslational Modifications ◽

Posttranslational Modification ◽

Drug Targets ◽

Viral Infections ◽

Interaction Analysis ◽

Human Interaction ◽

Host Cells ◽

Phosphorylation Sites ◽

Human Viruses ◽

Disordered Regions

Abstract In viruses, posttranslational modifications (PTMs) are essential for their life cycle. Recognizing viral PTMs is very important for a better understanding of the mechanism of viral infections and finding potential drug targets. However, few studies have investigated the roles of viral PTMs in virus–human interactions using comprehensive viral PTM datasets. To fill this gap, we developed the first comprehensive viral posttranslational modification database (VPTMdb) for collecting systematic information of PTMs in human viruses and infected host cells. The VPTMdb contains 1240 unique viral PTM sites with 8 modification types from 43 viruses (818 experimentally verified PTM sites manually extracted from 150 publications and 422 PTMs extracted from SwissProt) as well as 13 650 infected cells’ PTMs extracted from seven global proteomics experiments in six human viruses. The investigation of viral PTM sequences motifs showed that most viral PTMs have the consensus motifs with human proteins in phosphorylation and five cellular kinase families phosphorylate more than 10 viral species. The analysis of protein disordered regions presented that more than 50% glycosylation sites of double-strand DNA viruses are in the disordered regions, whereas single-strand RNA and retroviruses prefer ordered regions. Domain–domain interaction analysis indicating potential roles of viral PTMs play in infections. The findings should make an important contribution to the field of virus–human interaction. Moreover, we created a novel sequence-based classifier named VPTMpre to help users predict viral protein phosphorylation sites. VPTMdb online web server (http://vptmdb.com:8787/VPTMdb/) was implemented for users to download viral PTM data and predict phosphorylation sites of interest.

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AMP-activated protein kinase (AMPK) positively regulates osteoblast differentiation via induction of Dlx5-dependent Runx2 expression in MC3T3E1 cells

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2010.12.099 ◽

2011 ◽

Vol 404 (4) ◽

pp. 1004-1009 ◽

Cited By ~ 54

Author(s):

Won Gu Jang ◽

Eun Jung Kim ◽

Kkot-Nim Lee ◽

Hye-Ju Son ◽

Jeong-Tae Koh

Keyword(s):

Protein Kinase ◽

Osteoblast Differentiation ◽

Runx2 Expression ◽

Amp Activated Protein Kinase

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Protective Effects of Pretreatment with Quercetin Against Lipopolysaccharide-Induced Apoptosis and the Inhibition of Osteoblast Differentiation via the MAPK and Wnt/β-Catenin Pathways in MC3T3-E1 Cells

Cellular Physiology and Biochemistry ◽

10.1159/000481978 ◽

2017 ◽

Vol 43 (4) ◽

pp. 1547-1561 ◽

Cited By ~ 25

Author(s):

Chun Guo ◽

Rui-Juan Yang ◽

Ke Jang ◽

Xiao-ling Zhou ◽

Yu-zhen Liu

Keyword(s):

Cytochrome C ◽

Osteoblast Differentiation ◽

Caspase 3 ◽

Quantitative Polymerase Chain Reaction ◽

Bone Diseases ◽

Cell Counting ◽

Dependent Manner ◽

Protective Effects ◽

Expression Levels ◽

Induced Apoptosis

Background/Aims: Quercetin, a flavonoid found in onions and other vegetables, has potential inhibitory effects on bone resorption in vivo and in vitro. In our previous study, we found that quercetin treatment reversed lipopolysaccharide (LPS)-induced inhibition of osteoblast differentiation through the mitogen-activated protein kinase (MAPK) pathway in MC3T3-E1 cells. In this study, we investigated the underlying mechanisms of pretreatment with quercetin on apoptosis and the inhibition of osteoblast differentiation in MC3T3-E1 cells induced by LPS. Methods: MC3T3-E1 osteoblasts were treated with quercetin for 2 h; cells were then incubated with LPS in the presence of quercetin for the indicated times. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay, and cell apoptosis was evaluated using Hoechst 33258 staining. The mRNA expression levels of osteoblast-specific genes, Bax and caspase-3 were determined by real-time quantitative polymerase chain reaction (qPCR). Protein levels of osteoblast-specific genes, caspase-3, Bax, cytochrome c, Bcl-2, Bcl-XL, phosphorylated MAPKs and Wnt/β-catenin were measured using Western blot assays. The MAPK and Wnt/β-catenin signalling pathways were blocked prior to pretreatment with quercetin. Results: Pretreatment with quercetin significantly restored LPS-suppressed bone mineralization and the mRNA and protein expression levels of osteoblast-specific genes such as Osterix (OSX), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OCN) in a dose-dependent manner. Pretreatment with quercetin also inhibited osteoblast apoptosis, significantly restored the down-regulated expression of Bcl-2 and Bcl-XL and decreased the upregulated expression of caspase-3, Bax, and cytochrome c in MC3T3-E1 cells induced by LPS. Furthermore, pretreatment with quercetin not only decreased the abundance of phosphorylated p38 MAPK and increased the abundance of phosphorylated extracellular signal regulated kinase (ERK), but also triggered the Wnt/β-catenin pathway through enhancing expression of Wnt3 and β-catenin. Pretreatment with MAPK inhibitors or the Wnt/β-catenin inhibitor XAV939 blocked the protective effects of quercetin against LPS-induced apoptosis and the inhibition of osteoblast differentiation. Conclusions: Our findings suggest that pretreatment with quercetin may be a potential drug for preventing abnormal human bone loss induced by LPS in bacteria-induced bone diseases.

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A Systems Approach Identifies Key Regulators of HPV-Positive Cervical Cancer

10.1101/2020.05.12.20099424 ◽

2020 ◽

Cited By ~ 1

Author(s):

Musalula Sinkala ◽

Mildred Zulu ◽

Panji Nkhoma ◽

Doris Kafita ◽

Ephraim Zulu ◽

...

Keyword(s):

Cervical Cancer ◽

Regulatory Network ◽

Drug Targets ◽

Immune Cell ◽

Systems Approach ◽

Cell Signalling ◽

Hpv Infection ◽

Regulatory Pathways ◽

Cervical Cancers ◽

Functional Pathway

Cervical cancer has remained the most prevalent and lethal malignancy among women worldwide and accounted for over 250,000 deaths in 2019. Nearly ninety-five per cent of cervical cancer cases are associated with persistent infection with high-risk Human Papillomavirus (HPV), and seventy per cent of these are associated with viral integration in the host genome. HPV-infection imparts specific changes in the regulatory network of infected cancer cells that are of diagnostic, prognostic and therapeutic importance. Here, we conducted a systems-level analysis of the regulatory network changes, and the associated regulatory proteins thereof, in HPV-positive cervical cancer. We applied functional pathway analysis to show that HPV-positive cancers are characterised by perturbations of numerous cellular processes, predominantly in those linked to the cell cycle, mitosis, cytokine and immune cell signalling. Using computational predictions, we revealed that HPV-positive cervical cancers are regulated by transcription factors including, SOX2, E2F, NANOG, OCT4, and MYC, which control various processes such as the renewal of cancer stem cells, and the proliferation and differentiation of tumour cells. Through the analysis of upstream regulatory kinases, we identified the mitogen-activated protein kinases; among others, MAPK1, MAPK3 and MAPK8, and the cyclin-dependent kinases; among others, CDK1, CDK2 and CD4, as the key kinases that control the biological processes in HPV-positive cervical cancers. Taken together, we uncover a landscape of the key regulatory pathways and proteins in HPV-positive cervical cancers, all of which may provide attractive drug targets for future therapeutics.

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miR-206 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells by targetting glutaminase

Bioscience Reports ◽

10.1042/bsr20181108 ◽

2019 ◽

Vol 39 (3) ◽

Cited By ~ 15

Author(s):

Ying Chen ◽

Yu-Run Yang ◽

Xiao-Liang Fan ◽

Peng Lin ◽

Huan Yang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Osteoblast Differentiation ◽

Bone Diseases ◽

Glutamine Metabolism ◽

Mrna Levels ◽

Marrow Mesenchymal Stem Cells ◽

Osteogenic Induction

AbstractOsteoblast-mediated bone formation is a complex process involving various pathways and regulatory factors, including cytokines, growth factors, and hormones. Investigating the regulatory mechanisms behind osteoblast differentiation is important for bone regeneration therapy. miRNAs are known as important regulators, not only in a variety of cellular processes, but also in the pathogenesis of bone diseases. In the present study, we investigated the potential roles of miR-206 during osteoblast differentiation. We report that miR-206 expression was significantly down-regulated in human bone marrow mesenchymal stem cells (BMSCs) at days 7 and 14 during osteogenic induction. Furthermore, miR-206 overexpressing BMSCs showed attenuated alkaline phosphatase (ALP) activity, Alizarin Red staining, and osteocalcin secretion. The mRNA levels of osteogenic markers, Runx2 and Osteopontin (OPN), were significantly down-regulated in miR-206 overexpressing BMSCs. We observed that significantly increased glutamine uptake at days 7 and 14 during the osteogenic induction and inhibition of glutamine metabolism by knocking down glutaminase (GLS)-suppressed osteogenic differentiation of BMSCs. Here, we discover that miR-206 could directly bind to the 3′-UTR region of GLS mRNA, resulting in suppressed GLS expression and glutamine metabolism. Finally, restoration of GLS in miR-206 overexpressing BMSCs led to recovery of glutamine metabolism and osteogenic differentiation. In summary, these results reveal a new insight into the mechanisms of the miR-206-mediated osteogenesis through regulating glutamine metabolism. Our study may contribute to the development of therapeutic agents against bone diseases.

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Cbfa1 is required for epithelial-mesenchymal interactions regulating tooth development in mice

Development ◽

10.1242/dev.126.13.2911 ◽

1999 ◽

Vol 126 (13) ◽

pp. 2911-2920 ◽

Cited By ~ 8

Author(s):

R.N. D'Souza ◽

T. Aberg ◽

J. Gaikwad ◽

A. Cavender ◽

M. Owen ◽

...

Keyword(s):

Bone Formation ◽

Osteoblast Differentiation ◽

Tooth Development ◽

Enamel Organ ◽

Molecular Characteristics ◽

Cleidocranial Dysplasia ◽

Autosomal Dominant Disorder ◽

Tooth Formation ◽

Dental Epithelium

Osteoblasts and odontoblasts, cells that are responsible for the formation of bone and dentin matrices respectively, share several molecular characteristics. Recently, Cbfa1 was shown to be a critical transcriptional regulator of osteoblast differentiation. Mutations in this gene cause cleidocranial dysplasia (CCD), an autosomal dominant disorder in humans and mice characterized by defective bone formation. CCD also results in dental defects that include supernumerary teeth and delayed eruption of permanent dentition. The dental abnormalities in CCD suggest an important role for this molecule in the formation of dentition. Here we describe results of studies aimed at understanding the functions of Cbfa1 in tooth formation. RT-PCR and in situ hybridization analyses show that Cbfa1 has a unique expression pattern in dental mesenchyme from the bud to early bell stages during active epithelial morphogenesis. Unlike that observed in osteoblast differentiation, Cbfa1 is downregulated in fully differentiated odontoblasts and is surprisingly expressed in ectodermally derived ameloblasts during the maturation phase of enamel formation. The role of Cbfa1 in tooth morphogenesis is further illustrated by the misshapen and severely hypoplastic tooth organs in Cbfa1−/− mice. These tooth organs lacked overt odontoblast and ameloblast differentiation and normal dentin and enamel matrices. Epithelial-mesenchymal recombinants demonstrate that dental epithelium regulates mesenchymal Cbfa1 expression during the bud and cap stages and that these effects are mimicked by the FGFs but not by the BMPs as shown by our bead implantation assays. We propose that Cbfa1 regulates the expression of molecules in mesenchyme that act reciprocally on dental epithelium to control its growth and differentiation. Taken together, our data indicate a non-redundant role for Cbfa1 in tooth development that may be distinct from that in bone formation. In odontogenesis, Cbfa1 is not involved in the early signaling networks regulating tooth initiation and early morphogenesis but regulates key epithelial-mesenchymal interactions that control advancing morphogenesis and histodifferentiation of the epithelial enamel organ.

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The Effect of Alendronate on Proteome of Hepatocellular Carcinoma Cell Lines

International Journal of Proteomics ◽

10.1155/2014/532953 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 9

Author(s):

Amber Ilyas ◽

Zehra Hashim ◽

Nadia Naeem ◽

Kanwal Haneef ◽

Shamshad Zarina

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Lines ◽

Drug Targets ◽

Carcinoma Cell ◽

Control Cell ◽

Mva Pathway ◽

Regulatory Pathways ◽

Skeletal Complications ◽

Life Threatening ◽

Hepatocellular Carcinoma Cell

Cancer is a life threatening disorder effecting 11 million people worldwide annually. Among various types of cancers, Hepatocellular carcinoma (HCC) has a higher rate of mortality and is the fifth leading cause of cancer related deaths around the world. Many chemotherapeutic drugs have been used for the treatment of HCC with many side effects. These drugs are inhibitors of different cell regulatory pathways. Mevalonate (MVA) pathway is an important cellular cascade vital for cell growth. A variety of inhibitors of MVA pathway have been reported for their anticancerous activity. Bisphosphonates (BPs) are members of a family involved in the treatment of skeletal complications. In recent years, their anticancer potential has been highlighted. Current study focuses on exploring the effects of alendronate (ALN), a nitrogen containing BP, on hepatocellular carcinoma cell line using genomic and proteomics approach. Our results identified ten differentially expressed proteins, of which five were up regulated and five were down regulated in ALN treated cells. Furthermore, we also performed gene expression analysis in treated and control cell lines. The study may help in understanding the molecular mechanism involved in antitumor activity of ALN, identification of possible novel drug targets, and designing new therapeutic strategies for HCC.

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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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