scholarly journals Neu5Ac Induces Human Dental Pulp Stem Cell Osteo-/Odontoblastic Differentiation by Enhancing MAPK/ERK Pathway Activation

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Changzhou Li ◽  
Xinghuan Xie ◽  
Zhongjun Liu ◽  
Jianhua Yang ◽  
Daming Zuo ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Sialic Acid ◽  
Dental Pulp ◽  
Fluorescent Microscopy ◽  
Mapk Signaling ◽  
Permanent Teeth ◽  
Erk Pathway ◽  
Neuraminidase Treatment ◽  
Odontoblastic Differentiation

Dental pulp stem cells (DPSCs) must undergo odontoblastic differentiation in order to facilitate the process of dentin-pulp complex repair. Herein, we sought to explore the ability of Neu5Ac (one form of sialic acid) to influence DPSC osteo-/odontoblastic differentiation via modulating mitogen-activated protein kinase (MAPK) signaling. Methodology. DPSCs were isolated from human third permanent teeth and were grown in vitro. Fluorescent microscopy was used to detect the existence of sialic acid on the DPSC membrane. Following the treatment of different concentrations of Neu5Ac and removing sialic acid from the cell surface by neuraminidase, the osteo-/odontoblastic differentiation of these cells was evaluated via mineralization, alkaline phosphatase, and in vivo assays. In addition, the expression of genes related to osteo-/odontoblastic differentiation and MAPK signaling at different stages of this differentiation process was analyzed in the presence or absence of Neu5Ac. Results. The existence of sialic acid on the DPSC membrane was confirmed by fluorescent microscopy, and the ability of osteo-/odontoblastic differentiation was decreased after removing sialic acid by neuraminidase. Treatment of DPSCs with Neu5Ac (0.1 mM or 1 mM) significantly enhanced their mineralization ability and alkaline phosphatase activity. The expression levels of DMP1, DSPP, BSP, and RUNX2 were also increased. Treatment of nude mice with ManNAc (the prerequisite form of Neu5Ac) also enhanced DPSC mineralization activity in vivo. Furthermore, Neu5Ac treatment enhanced p-ERK expression in DPSCs, while ERK pathway inhibition disrupted the ability of Neu5Ac to enhance the osteo-/odontoblastic differentiation of these cells. Conclusions. Neu5Ac can promote DPSC osteo-/odontoblastic differentiation through a process associated with the modulation of the ERK signaling pathway activity.

17β-Estradiol induces odontoblastic differentiation via activation of the c-Src/MAPK pathway in human dental pulp cells

2015 ◽  
Vol 93 (6) ◽  
pp. 587-595 ◽  
Author(s):  
Su Mi Woo ◽  
Kyung Joo Seong ◽  
Sang Jin Oh ◽  
Hong Ju Park ◽  
Sun Hun Kim ◽  
...  
Keyword(s):  
Dental Pulp ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Dental Pulp Cells ◽  
Human Dental Pulp Cells ◽  
Odontoblastic Differentiation ◽  
Alp Activity ◽  
Human Dental Pulp ◽  
Pulp Cells ◽  
17Β Estradiol

The present study is aimed at investigating the effects of the exogenous estrogen 17β-estradiol (E2) on odontoblastic differentiation in human dental pulp cells (HDPCs) immotalized with hTERT gene and their molecular mechanism. Proliferation was detected by BrdU assay, and odontoblast differentiation induction was evaluated by the expression of dentin sialophosphoprotein (DSPP), dentin sialoprotein (DSP) and dentin matrix protein1 (DMP1), and alkaline phosphatase (ALP) activity and mineralization. Estrogen receptor-α (ER-α), c-Src, and mitogen-activated protein kinases (MAPKs) were examined and their inhibitors were used to determine the roles on odontogenic induction. E2 significantly promoted the HDPC proliferation, which was mediated by extracellular signal-related kinase 1/2. E2 upregulated DSPP, DSP, and DMP1 as the odontogenic differentiation markers and enhanced ALP activity and mineralization. E2 increased phosphorylation of ER-α and fulvestrant, an ER downregulator, significantly downregulated DSPP, DMP1, and DSP induced by E2. Moreover, E2 treatment activated c-Src and MAPKs upon odontogenic induction, whereas chemical inhibition of c-Src and MAPKs decreased expression of DSPP, DMP1, and DSP and mineralization augmented by E2. Moreover, fulvestrant reduced E2-induced phosphorylation of c-Src and MAPK and inhibition of c-Src by PP2 attenuated activation of MAPKs during E2-induced odontoblastic differentiation. Taken together, these results indicated that E2 stimulates odontoblastic differentiation of HDPCs via coordinated regulation of ER-α, c-Src, and MAPK signaling pathways, which may play a key role in the regeneration of dentin.

scholarly journals LncRNA H19 Promotes Odontoblastic Differentiation of Human Dental Pulp Stem Cells by Regulating miR-140-5p and BMP-2/FGF9

2020 ◽  
Author(s):  
Jialin Zhong ◽  
Xinran Tu ◽  
Yuanyuan Kong ◽  
Liyang Guo ◽  
Baishun Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dental Pulp ◽  
Dental Pulp Stem Cells ◽  
Biological Role ◽  
Luciferase Reporter ◽  
Qrt Pcr ◽  
Odontoblastic Differentiation ◽  
Human Dental Pulp

Abstract Background: Increasing evidence has revealed that long non-coding RNAs (lncRNAs) exert critical roles in biological mineralization. As a critical process for dentin formation, odontoblastic differentiation is regulated by complex signaling networks. The present study aimed to investigate the biological role and regulatory mechanisms of lncRNA-H19 (H19) in regulating the odontoblastic differentiation of human dental pulp stem cells (hDPSCs). Methods: We performed lncRNA microarray assay to reveal the expression patterns of lncRNAs involved in odontoblastic differentiation. H19 was identified and verified by qRT-PCR. The gain- and loss-of-function studies were performed to investigate the biological role of H19 in regulating odontoblastic differentiation of hDPSCs in vitro and in vivo. Odontoblastic differentiation was evaluated through qRT-PCR, Western blot and Alizarin Red S staining. Bioinformatics analysis identified that H19 could directly interact with miR-140-5p, which was further verified by luciferase reporter assay. After overexpression of miR-140-5p in hDPSCs, odontoblastic differentiation was determined. Moreover, the potential target genes of miR-140-5p were investigated and the biological functions of BMP-2 and FGF9 in hDPSCs were verified. Co-transfection experiments were conducted to validate miR-140-5p was involved in H19-mediated odontoblastic differentiation in hDPSCs.Results: The expression of H19 was significantly up-regulated in hDPSCs undergoing odontoblastic differentiation. Overexpression of H19 stimulated odontoblastic differentiation in vitro and in vivo, whereas down-regulation of H19 revealed the opposite effect. H19 binds directly to miR-140-5p and overexpression of miR-140-5p inhibited odontoblastic differentiation of hDPSCs. H19 acted as a miR-140-5p sponge, resulting in regulated the expression of BMP-2 and FGF9. Overexpression of H19 abrogated the inhibitory effect of miR-140-5p on odontoblastic differentiation.Conclusion: Our data revealed that H19 plays a positive regulatory role in odontoblastic differentiation of hDPSCs through miR-140-5p/BMP-2/FGF9 axis, suggesting that H19 may be a stimulatory regulator of odontogenesis.

Cell Surface Sialoglycopeptide Metabolism and Surface Glycosyl Transferase Activity in the Ehrlich Ascites Cell

1975 ◽  
Vol 53 (8) ◽  
pp. 895-902 ◽  
Author(s):  
D. Irwin ◽  
T. P. Anastassiades
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Transferase Activity ◽  
Intact Cells ◽  
Neuraminidase Treatment ◽  
Glycosyl Transferase ◽  
Ehrlich Ascites ◽  
Ehrlich Ascites Cell ◽  
Almost All

1. The in vivo incorporation of radioactivity from [3H]glucosamine into a trypsin labile, cell surface sialoglycopeptide fraction (SGP) of Ehrlich ascites cells was studied in the presence and absence of puromycin pretreatment. The results indicated a much more complete inhibition of incorporation into the surface SGP than in the average intracellular acid insoluble glycoproteins. No evidence of turnover of the carbohydrate portion of the surface SGP independent of protein synthesis could be obtained.2. However, when intact cells were incubated with labelled uridine 5′-diphosphate – N-acetyl glucosamine or cytidine 5′-monophosphate (CMP) – sialic acid there was some incorporation largely into acid insoluble material, suggesting the presence of glycosyl transferase activity at the surface. Further evidence for surface activity was obtained when neuraminidase pretreatment of intact cells stimulated incorporation of labelled CMP – sialic acid sixfold and almost all of the incorporated counts could be released by subsequent neuraminidase treatment. Furthermore, a much greater proportion of the incorporated counts could be released by papain than by trypsin treatment of the intact cells. These results suggest that the surface acceptor for exogenously added CMP – sialic acid is not identical to the endogenously synthesized trypsin labile surface SGP.

scholarly journals Binding of the Streptococcus gordonii DL1 Surface Protein Hsa to the Host Cell Membrane Glycoproteins CD11b, CD43, and CD50

2008 ◽  
Vol 76 (10) ◽  
pp. 4686-4691 ◽  
Author(s):  
Yumiko Urano-Tashiro ◽  
Ayako Yajima ◽  
Eizo Takashima ◽  
Yukihiro Takahashi ◽  
Kiyoshi Konishi
Keyword(s):  
Sialic Acid ◽  
Infective Endocarditis ◽  
Surface Protein ◽  
Oral Streptococci ◽  
Streptococcus Gordonii ◽  
Membrane Glycoproteins ◽  
Host Cell Membrane ◽  
Neuraminidase Treatment ◽  
Sialic Acid Binding

ABSTRACTInfective endocarditis is frequently attributed to oral streptococci. The mechanisms of pathogenesis, however, are not well understood, although interaction between streptococci and phagocytes are thought to be very important. A highly expressed surface component ofStreptococcus gordonii, Hsa, which has sialic acid-binding activity, contributes to infective endocarditis in vivo. In the present study, we found thatS. gordoniiDL1 binds to HL-60 cells differentiated into monocytes, granulocytes, and macrophages. Using a glutathioneS-transferase (GST) fusion to the NR2 domain, which is the sialic acid-binding region of Hsa, we confirmed that the Hsa NR2 domain also binds to differentiated HL-60 cells. To identify which sialoglycoproteins on the surface of differentiated HL-60 cells are receptors for Hsa, intrinsic membrane proteins were assessed by bacterial overlay and far-Western blotting.S. gordoniiDL1 adhered to 100- to 150-kDa proteins, a reaction that was abolished by neuraminidase treatment. These sialoglycoproteins were identified as CD11b, CD43, and CD50 by GST pull-down assay and immunoprecipitation with each specific monoclonal antibody. These data suggest thatS. gordoniiDL1 Hsa specifically binds to three glycoproteins as receptors and that this interaction may be the initial bacterial binding step in infective endocarditis by oral streptococci.

scholarly journals Inverse and reciprocal regulation of p53/p21 and Bmi-1 modulates vasculogenic differentiation of dental pulp stem cells

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Zhaocheng Zhang ◽  
Min Oh ◽  
Jun-Ichi Sasaki ◽  
Jacques E. Nör
Keyword(s):  
Stem Cells ◽  
Dental Pulp ◽  
Smooth Muscle Actin ◽  
Dental Pulp Stem Cells ◽  
Permanent Teeth ◽  
Vascular Endothelial ◽  
P53 Expression ◽  
Immunodeficient Mice

AbstractDental pulp stem cells (DPSC) are capable of differentiating into vascular endothelial cells. Although the capacity of vascular endothelial growth factor (VEGF) to induce endothelial differentiation of stem cells is well established, mechanisms that maintain stemness and prevent vasculogenic differentiation remain unclear. Here, we tested the hypothesis that p53 signaling through p21 and Bmi-1 maintains stemness and inhibits vasculogenic differentiation. To address this hypothesis, we used primary human DPSC from permanent teeth and Stem cells from Human Exfoliated Deciduous (SHED) teeth as models of postnatal mesenchymal stem cells. DPSC seeded in biodegradable scaffolds and transplanted into immunodeficient mice generated mature human blood vessels invested with smooth muscle actin-positive mural cells. Knockdown of p53 was sufficient to induce vasculogenic differentiation of DPSC (without vasculogenic differentiation medium containing VEGF), as shown by increased expression of endothelial markers (VEGFR2, Tie-2, CD31, VE-cadherin), increased capillary sprouting in vitro; and increased DPSC-derived blood vessel density in vivo. Conversely, induction of p53 expression with small molecule inhibitors of the p53-MDM2 binding (MI-773, APG-115) was sufficient to inhibit VEGF-induced vasculogenic differentiation. Considering that p21 is a major downstream effector of p53, we knocked down p21 in DPSC and observed an increase in capillary sprouting that mimicked results observed when p53 was knocked down. Stabilization of ubiquitin activity was sufficient to induce p53 and p21 expression and reduce capillary sprouting. Interestingly, we observed an inverse and reciprocal correlation between p53/p21 and the expression of Bmi-1, a major regulator of stem cell self-renewal. Further, direct inhibition of Bmi-1 with PTC-209 resulted in blockade of capillary-like sprout formation. Collectively, these data demonstrate that p53/p21 functions through Bmi-1 to prevent the vasculogenic differentiation of DPSC.

scholarly journals LncRNA H19 promotes odontoblastic differentiation of human dental pulp stem cells by regulating miR-140-5p and BMP-2/FGF9

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jialin Zhong ◽  
Xinran Tu ◽  
Yuanyuan Kong ◽  
Liyang Guo ◽  
Baishun Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dental Pulp ◽  
Dental Pulp Stem Cells ◽  
Biological Role ◽  
Luciferase Reporter ◽  
Qrt Pcr ◽  
Odontoblastic Differentiation ◽  
Human Dental Pulp

Abstract Background Increasing evidence has revealed that long non-coding RNAs (lncRNAs) exert critical roles in biological mineralization. As a critical process for dentin formation, odontoblastic differentiation is regulated by complex signaling networks. The present study aimed to investigate the biological role and regulatory mechanisms of lncRNA-H19 (H19) in regulating the odontoblastic differentiation of human dental pulp stem cells (hDPSCs). Methods We performed lncRNA microarray assay to reveal the expression patterns of lncRNAs involved in odontoblastic differentiation. H19 was identified and verified as a critical factor by qRT-PCR. The gain- and loss-of-function studies were performed to investigate the biological role of H19 in regulating odontoblastic differentiation of hDPSCs in vitro and in vivo. Odontoblastic differentiation was evaluated through qRT-PCR, Western blot, and Alizarin Red S staining. Bioinformatics analysis identified that H19 could directly interact with miR-140-5p, which was further verified by luciferase reporter assay. After overexpression of miR-140-5p in hDPSCs, odontoblastic differentiation was determined. Moreover, the potential target genes of miR-140-5p were investigated and the biological functions of BMP-2 and FGF9 in hDPSCs were verified. Co-transfection experiments were conducted to validate miR-140-5p was involved in H19-mediated odontoblastic differentiation in hDPSCs. Results The expression of H19 was significantly upregulated in hDPSCs undergoing odontoblastic differentiation. Overexpression of H19 stimulated odontoblastic differentiation in vitro and in vivo, whereas downregulation of H19 revealed the opposite effect. H19 binds directly to miR-140-5p and overexpression of miR-140-5p inhibited odontoblastic differentiation of hDPSCs. H19 acted as a miR-140-5p sponge, resulting in regulated the expression of BMP-2 and FGF9. Overexpression of H19 abrogated the inhibitory effect of miR-140-5p on odontoblastic differentiation. Conclusion Our data revealed that H19 plays a positive regulatory role in odontoblastic differentiation of hDPSCs through miR-140-5p/BMP-2/FGF9 axis, suggesting that H19 may be a stimulatory regulator of odontogenesis.

Abstract 1207: In Vivo Molecular Imaging Links Macrophage Burden and Microcalcifications in Early Atherosclerosis

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Elena Aikawa ◽  
Matthias Nahrendorf ◽  
Jose-Luiz Figueiredo ◽  
Filip K Swirski ◽  
Timur Shtatland ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Molecular Imaging ◽  
Near Infrared ◽  
Plaque Rupture ◽  
Fluorescent Microscopy ◽  
Arterial Calcification ◽  
Target Cells ◽  
Osteogenic Activity ◽  
Nirf Imaging

Backround Microcalcifications may provoke plaque rupture leading to acute cardiovascular events. However, the mechanisms that initiate arterial calcification remain obscure. Methods and Results We tested the hypothesis that inflammation promotes osteogenesis in atherosclerotic plaques using in vivo molecular imaging in apoE −/− mice (n=40). A bisphosphonate-derivatized near-infrared fluorescent (NIRF) imaging agent (750 nm) visualized osteogenic activity that was otherwise undetectable by X-ray computed tomography. Flow cytometry validated target cells as osteoblasts. A spectrally distinct NIRF nanoparticle (680 nm) was coinjected to simultaneously image macrophages. Fluorescence reflectance mapping associated osteogenic activity with inflammation in aortas of apoE −/− mice. Intravital dual-channel fluorescent microscopy further monitored osteogenic changes in carotid plaques at 20 and 30 weeks of age and revealed that both macrophage burden and osteogenesis concomitantly increased during plaque progression and decreased after statin treatment (Figure ) . Fluorescence microscopy on cryosections colocalized NIRF osteogenic signals with alkaline phosphatase activity, bone-regulating proteins and hydroxyapatite nanocrystals detected by electron microscopy, while von Kossa staining showed no evidence of calcification. Real-time RT-PCR revealed that macrophage-conditioned media induced alkaline phosphatase mRNA expression in vascular smooth muscle cells. Conclusion This serial in vivo study detects osteogenic activity within macrophage-rich plaques, offering a cellular resolution tool to identify preclinical microcalcifications.

scholarly journals Investigation of inhibition effect of daidzein on osteosarcoma cells based on experimental validation and systematic pharmacology analysis

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12072
Author(s):  
Yufan Zhu ◽  
Zhiqiang Yang ◽  
Yuanlong Xie ◽  
Min Yang ◽  
Yufeng Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Mapk Signaling ◽  
Osteosarcoma Cell ◽  
Erk Pathway ◽  
Osteosarcoma Cells ◽  
Antitumor Effects ◽  
Xenograft Mice ◽  
And Migration

Objective This study aims to explore the effect of daidzein, which is a natural isoflavone compound mainly extracted from soybeans, on osteosarcoma and the potential molecular mechanism. Material and Methods 143B and U2OS osteosarcoma cells were treated with gradient concentrations of daidzein, and MTT assay was used to determine the cell proliferation capacity and IC50. Hoechst 33342 staining and Annexin V-FITC/PI detection were used to determine apoptosis. Cell cycle was analyzed by flow cytometry, and migration ability were detected by transwell assays and scratch wound assay. An osteosarcoma xenograft mice model was applied to investigate the effect of daidzein on osteosarcoma in vivo. Systematic pharmacology and molecular modeling analysis were applied to predict the target of daidzein to osteosarcoma, and the target Src was verified by western blotting. We also observed the effect of daidzein on cell proliferation and apoptosis of Src-overexpressing osteosarcoma cells. Results In vitro, daidzein significantly inhibited 143B and U2OS osteosarcoma cell proliferation and migration, and induced cell cycle arrest. In vivo, daidzein exerts antitumor effects in osteosarcoma xenograft mice. After systematic screening and analysis, Src-MAPK signaling pathway was predicted as the highest-ranked pathway. Western blot demonstrated that daidzein inhibited phosphorylation of the Src-ERK pathway in osteosarcoma cells. Also, overexpression of Src could partially reverse the inhibitory effects of daidzein on osteosarcoma cell proliferation. Conclusion Daidzein exerts an antitumor effect on osteosarcoma, and the mechanism may be through the Src-ERK pathway.

scholarly journals The Viral σ1 Protein and Glycoconjugates Containing α2-3-Linked Sialic Acid Are Involved in Type 1 Reovirus Adherence to M Cell Apical Surfaces

2003 ◽  
Vol 77 (14) ◽  
pp. 7964-7977 ◽  
Author(s):  
Anna Helander ◽  
Katherine J. Silvey ◽  
Nicholas J. Mantis ◽  
Amy B. Hutchings ◽  
Kartik Chandran ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Apical Cell ◽  
M Cells ◽  
M Cell ◽  
Neuraminidase Treatment ◽  
Apical Cell Membrane ◽  
Follicle Associated Epithelium ◽  
Type 3

ABSTRACT Type 1 reoviruses invade the intestinal mucosa of mice by adhering selectively to M cells in the follicle-associated epithelium and then exploiting M cell transport activity. The purpose of this study was to identify the apical cell membrane component and viral protein that mediate the M cell adherence of these viruses. Virions and infectious subviral particles of reovirus type 1 Lang (T1L) adhered to rabbit M cells in Peyer's patch mucosal explants and to tissue sections in an overlay assay. Viral adherence was abolished by pretreatment of sections with periodate and in the presence of excess sialic acid or lectins MAL-I and MAL-II (which recognize complex oligosaccharides containing sialic acid linked α2-3 to galactose). The binding of T1L particles to polarized human intestinal (Caco-2BBe) cell monolayers was correlated with the presence of MAL-I and MAL-II binding sites, blocked by excess MAL-I and -II, and abolished by neuraminidase treatment. Other type 1 reovirus isolates exhibited MAL-II-sensitive binding to rabbit M cells and polarized Caco-2BBe cells, but type 2 or type 3 isolates including type 3 Dearing (T3D) did not. In assays using T1L-T3D reassortants and recoated viral cores containing T1L, T3D, or no σ1 protein, MAL-II-sensitive binding to rabbit M cells and polarized Caco-2BBe cells was consistently associated with the T1L σ1. MAL-II-recognized oligosaccharide epitopes are not restricted to M cells in vivo, but MAL-II immobilized on virus-sized microparticles bound only to the follicle-associated epithelium and M cells. The results suggest that selective binding of type 1 reoviruses to M cells in vivo involves interaction of the type 1 σ1 protein with glycoconjugates containing α2-3-linked sialic acid that are accessible to viral particles only on M cell apical surfaces.

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