DOCSLIB.ORG

Impaired Osteoblast Differentiation in Annexin A2- and - A5-Deficient Cells

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Impaired Osteoblast Differentiation in Annexin A2- and - A5-Deficient Cells Impaired Osteoblast Differentiation in Annexin A2- and - A5-Deficient Cells Damian C. Genetos1, Alice Wong1, Thomas J. Weber2, Norman J. Karin2, Clare E. Yellowley1* 1 Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California Davis, Davis, CA, United States of America, 2 Systems Toxicology, Pacific Northwest National Laboratory, Richland, WA, United States of America Abstract Annexins are a class of calcium-binding proteins with diverse functions in the regulation of lipid rafts, inflammation, fibrinolysis, transcriptional programming and ion transport. Within bone, they are well-characterized as components of mineralizing matrix vesicles, although little else is known as to their function during osteogenesis. We employed shRNA to generate annexin A2 (AnxA2)- or annexin A5 (AnxA5)-knockdown pre-osteoblasts, and determined whether proliferation or osteogenic differentiation was altered in knockdown cells, compared to pSiren (Si) controls. We report that DNA content, a marker of proliferation, was significantly reduced in both AnxA2 and AnxA5 knockdown cells. Alkaline phosphatase expression and activity were also suppressed in AnxA2- or AnxA5-knockdown after 14 days of culture. The pattern of osteogenic gene expression was altered in knockdown cells, with Col1a1 expressed more rapidly in knock-down cells, compared to pSiren. In contrast, Runx2, Ibsp, and Bglap all revealed decreased expression after 14 days of culture. In both AnxA2- and AnxA5-knockdown, interleukin-induced STAT6 signaling was markedly attenuated compared to pSiren controls. These data suggest that AnxA2 and AnxA5 can influence bone formation via regulation of osteoprogenitor proliferation, differentiation, and responsiveness to cytokines in addition to their well-studied function in matrix vesicles. Citation: Genetos DC, Wong A, Weber TJ, Karin NJ, Yellowley CE (2014) Impaired Osteoblast Differentiation in Annexin A2- and -A5-Deficient Cells. PLoS ONE 9(9): e107482. doi:10.1371/journal.pone.0107482 Editor: Carlos M. Isales, Georgia Regents University, United States of America Received May 13, 2013; Accepted August 18, 2014; Published September 15, 2014 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by Award Number R03AR057547 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (DCG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] Introduction into increases in intracellular calcium and inducing gene transcription in osteoblasts [17]. With regards to the hematopoi- Annexins comprise a class of calcium-dependent, phospholipid- etic component of the skeleton, exogenous AnxA2 increases the binding proteins that are broadly expressed in eukaryotic cells. formation of human bone marrow multinucleated cells, TRAP- They are predominately localized within the cell, where they positive staining, and dentine resorption [18]. Certain of these mediate such cellular processes as exocytosis and endocytosis, effects occur indirectly, as AnxA2 increases pre-osteoclast prolif- membrane structure and generation of lipid rafts, formation or eration by increasing GM-CSF production from bone marrow regulation of ion channels, and cytokinesis. A subset of annexins stromal cells and activated T cells [19], and promotes ERK1/2- have extracellular roles, and participate in regulation of inflam- dependent RANKL secretion from bone marrow stromal cells mation, coagulation and fibrinolysis (reviewed in [1]). More [17,20,21]. Gillette and Nielsen-Preiss demonstrated that over- recently, they have been identified as key mediators in maintaining expression of AnxA2 in human osteosarcoma cells facilitates the endothelial and hematopoietic stem cells within the bone marrow terminal stages of osteogenic differentiation, specifically matrix niche [2,3] and as pivotal regulators of metastasis and adhesion of mineralization [22], although if AnxA2 exerted a role prior to prostate cancer cells within bone [4]. mineralization was not examined. Of the 12 Annexins expressed in mammals, Annexins A1, A2, While these data indicate a role for AnxA2 in matrix A4, A5, A6 and A7 are expressed within cells of the chondrogenic mineralization, whether either AnxA2 or AnxA5 have cell- and osteoblastic lineage [5–7]. To date, their function within these autonomous effects on processes occurring prior to mineraliza- cells has primarily focused upon a putative role in matrix tion–proliferation and osteogenic differentiation–remains unex- mineralization. AnxA5 is involved in endochondral ossification, amined. In this study, we examined the influence of depletion of and is sequentially expressed during vasculogenesis and formation AnxA2 or AnxA5 (AnxA2kd and AnxA5kd, respectively) upon the of the cartilage anlage [8,9]. During embryogenesis and post-natal proliferation and osteogenic differentiation of the pre-osteoblast skeletal development, AnxA2 and AnxA5 are present in matrix MC3T3-E1 cell line. Reduced expression of AnxA2 or AnxA5 vesicles secreted by hypertrophic chondrocytes and osteoblasts [10–15]. Similarly, Annexins A1, A4, and A7 are also found within decreased proliferation and altered the dynamic course of matrix vesicles from mineralizing osteoblasts [16]. However, little osteogenic differentiation compared to pSiren (Si) control cells. data exist as to whether, and when, AnxA2 or AnxA5 exert cell- Mechanistically, AnxA2kd and AnxA5kd each demonstrated autonomous roles in an osteoblast. We have reported that AnxA5 decreased responsiveness to the anti-inflammatory cytokine is involved in transducing a biophysical signal–fluid shear stress– interleukin 4 (IL-4), indicating that both AnxA2 and AnxA5 are PLOS ONE | www.plosone.org 1 September 2014 | Volume 9 | Issue 9 | e107482 Annexins in Osteoblast Differentiation required for maximal responsiveness. In total, these data Proliferation decreases and the dynamics of osteogenic demonstrate cell-autonomous roles for both AnxA2 and AnxA5 differentiation are disrupted in Annexin-deficient cells in proliferation of pre-osteoblasts, matrix maturation and miner- Having demonstrated significant reduction of AnxA2 and alization. AnxA5 mRNA and protein, we next examined whether alterations in AnxA2 or AnxA5 expression influenced pre-osteoblast prolif- Results eration. Total DNA content was significantly reduced in AnxA2kd (14% reduction) and in AnxA5kd (20% reduction) cells, relative to Annexin A2 and A5 expression in knockdown cell lines Si control (Figure 2A). Similar reductions in proliferation were Stable MC3T3-E1 cell lines deficient in AnxA2 and AnxA5 observed using Calcein-AM (reductions of 29% and 30%; expression were generated as described in Materials and Methods. Figure 2B) and Alamar Blue (18% and 20%; Figure 2C) assays There was a significant reduction (.80%) in mRNA AnxA2 in AnxA2kd and AnxA5kd, respectively. expression in kd cells compared to Si-transfected controls AnxA2 Annexins are implicated in matrix mineralization by nature of (Figure 1A), and there was no compensatory change in AnxA5 their presence in matrix vesicles isolated from osteoblasts and m in kd cells. Similarly, there was a significant RNA AnxA2 chondroblasts, although there is no data whether Annexins may reduction in mRNA in kd cells compared to Si AnxA5 AnxA5 have a role in earlier stages of osteogenic differentiations, control, and no effect of depletion upon mRNA AnxA5 AnxA2 specifically matrix formation or maturation. Thus, we examined expression (Figure 1B). Changes in Annexin expression were also the influence of AnxA2 or AnxA5 reduction upon markers of confirmed at the protein level by western immunoblot (Fig- osteogenic differentiation. Si control cells showed a dynamic ure 1C). Densitometric analysis relative to a-tubulin showed that pattern of gene expression associated with osteogenic differentia- AnxA2 protein expression in AnxA2kd was approximately 50% of tion (Col1a1, Runx2, Ibsp, Sp7, Spp1 and Bglap) [23]. For Si Si control (Figure 1D). AnxA5 protein expression was reduced by controls, initiation of osteogenesis at 0 days resulted in significant approximately 40% in AnxA5kd cells compared to Si control increases in the osteogenic transcription factors Runx2 and (Figure 1E). Osterix (Sp7) after 14 days in culture, and was maintained at 21 days of culture compared to day 0 controls (Figures 3A and B). A similar time course of Runx2 induction and level of expression was observed in both AnxA2kd and AnxA5kd cells (Figure 3A), although Sp7 expression decreased significantly at 21 days in culture compared to Si controls (Figure 3B). For each cell type, osteogenic differentiation increased Col1a1 expression, although induction expression occurred earlier in AnxA2kd and AnxA5kd cells compared to Si controls (Figure 3C), suggesting that the ordered process of osteogenic differentiation was subtly altered in AnxA2kd and AnxA5kd cells. Non-collagenous proteins implicat- ed in matrix maturation and ordered deposition of hydroxyapa- tite–bone sialoprotein (Ibsp) and osteocalcin (Bglap or Ocn)–each revealed altered patterns of expression in knockdown cells compared
Load more

Recommended publications
  • Functions of Osteocalcin in Bone, Pancreas, Testis, and Muscle
    International Journal of Molecular Sciences Review Functions of Osteocalcin in Bone, Pancreas, Testis, and Muscle Toshihisa Komori Basic and Translational Research Center for Hard Tissue Disease, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan; [email protected]; Tel.: +81-95-819-7637; Fax: +81-95-819-7638 Received: 17 September 2020; Accepted: 10 October 2020; Published: 12 October 2020 Abstract: Osteocalcin (Ocn), which is specifically produced by osteoblasts, and is the most abundant non-collagenous protein in bone, was demonstrated to inhibit bone formation and function as a hormone, which regulates glucose metabolism in the pancreas, testosterone synthesis in the testis, / / and muscle mass, based on the phenotype of Ocn− − mice by Karsenty’s group. Recently, Ocn− − mice were newly generated by two groups independently. Bone strength is determined by bone / quantity and quality. The new Ocn− − mice revealed that Ocn is not involved in the regulation of bone formation and bone quantity, but that Ocn regulates bone quality by aligning biological apatite (BAp) parallel to the collagen fibrils. Moreover, glucose metabolism, testosterone synthesis and / spermatogenesis, and muscle mass were normal in the new Ocn− − mice. Thus, the function of Ocn is the adjustment of growth orientation of BAp parallel to the collagen fibrils, which is important for bone strength to the loading direction of the long bone. However, Ocn does not play a role as a hormone in the pancreas, testis, and muscle. Clinically, serum Ocn is a marker for bone formation, and exercise increases bone formation and improves glucose metabolism, making a connection between Ocn and glucose metabolism.
    [Show full text]
  • Annexin A2 Flop-Out Mediates the Non-Vesicular Release of Damps/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions
    cells Article Annexin A2 Flop-Out Mediates the Non-Vesicular Release of DAMPs/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions Hayato Matsunaga 1,2,† , Sebok Kumar Halder 1,3,† and Hiroshi Ueda 1,4,* 1 Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521, Japan; [email protected] (H.M.); [email protected] (S.K.H.) 2 Department of Medical Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8523, Japan 3 San Diego Biomedical Research Institute, San Diego, CA 92121, USA 4 Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan * Correspondence: [email protected]; Tel.: +81-75-753-4536 † These authors contributed equally to this work. Abstract: Prothymosin alpha (ProTα) and S100A13 are released from C6 glioma cells under serum- free conditions via membrane tethering mediated by Ca2+-dependent interactions between S100A13 and p40 synaptotagmin-1 (Syt-1), which is further associated with plasma membrane syntaxin-1 (Stx-1). The present study revealed that S100A13 interacted with annexin A2 (ANXA2) and this interaction was enhanced by Ca2+ and p40 Syt-1. Amlexanox (Amx) inhibited the association between S100A13 and ANXA2 in C6 glioma cells cultured under serum-free conditions in the in situ proximity ligation assay. In the absence of Amx, however, the serum-free stress results in a flop-out of ANXA2 Citation: Matsunaga, H.; Halder, through the membrane, without the extracellular release. The intracellular delivery of anti-ANXA2 S.K.; Ueda, H. Annexin A2 Flop-Out antibody blocked the serum-free stress-induced cellular loss of ProTα, S100A13, and Syt-1.
    [Show full text]
  • Annexin A1 Is a New Functional Linker Between Actin Filaments and Phagosomes During Phagocytosis
    578 Research Article Annexin A1 is a new functional linker between actin filaments and phagosomes during phagocytosis Devang M. Patel1, Syed Furquan Ahmad1, Dieter G. Weiss1, Volker Gerke2 and Sergei A. Kuznetsov1,* 1Institute of Biological Sciences, Cell Biology and Biosystems Technology, University of Rostock, Albert-Einstein Straße 3, Rostock 18059, Germany 2Institute of Medical Biochemistry, Centre for Molecular Biology of Inflammation, University of Münster, Von-Esmarch-Straße 56, Münster 48149, Germany *Author for correspondence ([email protected]) Accepted 19 October 2010 Journal of Cell Science 124, 578-588 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.076208 Summary Remodelling of the actin cytoskeleton plays a key role in particle internalisation and the phagosome maturation processes. Actin- binding proteins (ABPs) are the main players in actin remodelling but the precise role of these proteins in phagocytosis needs to be clarified. Annexins, a group of ABPs, are known to be present on phagosomes. Here, we identified annexin A1 as a factor that binds to isolated latex bead phagosomes (LBPs) in the presence of Ca2+ and facilitates the F-actin–LBP interaction in vitro. In macrophages the association of endogenous annexin A1 with LBP membranes was strongly correlated with the spatial and temporal accumulation of F-actin at the LBP. Annexin A1 was found on phagocytic cups and around early phagosomes, where the F-actin was prominently concentrated. After uptake was completed, annexin A1, along with F-actin, dissociated from the nascent LBP surface. At later stages of phagocytosis annexin A1 transiently concentrated only around those LBPs that showed transient F-actin accumulation (‘actin flashing’).
    [Show full text]
  • 2812 Matrix Vesicles: Structure, Composition, Formation and Function in Ca
    [Frontiers in Bioscience 16, 2812-2902, June 1, 2011] Matrix vesicles: structure, composition, formation and function in calcification Roy E. Wuthier Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208 TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Morphology of matrix vesicles (MVs) 3.1. Conventional transmission electron microscopy 3.2. Cryofixation, freeze-substitution electron microscopy 3.3. Freeze-fracture studies 4. Isolation of MVs 4.1. Crude collagenase digestion methods 4.2. Non-collagenase dependent methods 4.3. Cell culture methods 4.4. Modified collagenase digestion methods 4.5. Other isolation methods 5. MV proteins 5.1. Early SDS-PAGE studies 5.2. Isolation and identification of major MV proteins 5.3. Sequential extraction, separation and characterization of major MV proteins 5.4. Proteomic characterization of MV proteins 6. MV-associated extracellular matrix proteins 6.1. Type VI collagen 6.2. Type X collagen 6.3. Proteoglycan link protein and aggrecan core protein 6.4. Fibrillin-1 and fibrillin-2 7. MV annexins – acidic phospholipid-dependent ca2+-binding proteins 7.1. Annexin A5 7.2. Annexin A6 7.3. Annexin A2 7.4. Annexin A1 7.5. Annexin A11 and Annexin A4 8. MV enzymes 8.1. Tissue-nonspecific alkaline phosphatase(TNAP) 8.1.1. Molecular structure 8.1.2. Amino acid sequence 8.1.3. 3-D structure 8.1.4. Disposition in the MV membrane 8.1.5. Catalytic properties 8.1.6. Collagen-binding properties 8.2. Nucleotide pyrophosphate phosphodiesterase (NPP1, PC1) 8.3. PHOSPHO-1 (Phosphoethanolamine/Phosphocholine phosphatase 8.4. Acid phosphatase 8.5.
    [Show full text]
  • Changes in the Cytosolic Proteome of Aedes Malpighian Tubules
    329 The Journal of Experimental Biology 212, 329-340 Published by The Company of Biologists 2009 doi:10.1242/jeb.024646 Research article Signaling to the apical membrane and to the paracellular pathway: changes in the cytosolic proteome of Aedes Malpighian tubules Klaus W. Beyenbach1,*, Sabine Baumgart2, Kenneth Lau1, Peter M. Piermarini1 and Sheng Zhang2 1Department of Biomedical Sciences, VRT 8004, Cornell University, Ithaca, NY 14853, USA and 2Proteomics and Mass Spectrometry Core Facility, 143 Biotechnology Building, Cornell University, Ithaca, NY 14853, USA *Author for correspondence (e-mail: [email protected]) Accepted 6 November 2008 Summary Using a proteomics approach, we examined the post-translational changes in cytosolic proteins when isolated Malpighian tubules of Aedes aegypti were stimulated for 1 min with the diuretic peptide aedeskinin-III (AK-III, 10–7 mol l–1). The cytosols of control (C) and aedeskinin-treated (T) tubules were extracted from several thousand Malpighian tubules, subjected to 2-D electrophoresis and stained for total proteins and phosphoproteins. The comparison of C and T gels was performed by gel image analysis for the change of normalized spot volumes. Spots with volumes equal to or exceeding C/T ratios of ±1.5 were robotically picked for in- gel digestion with trypsin and submitted for protein identification by nanoLC/MS/MS analysis. Identified proteins covered a wide range of biological activity. As kinin peptides are known to rapidly stimulate transepithelial secretion of electrolytes and water by Malpighian tubules, we focused on those proteins that might mediate the increase in transepithelial secretion. We found that AK- III reduces the cytosolic presence of subunits A and B of the V-type H+ ATPase, endoplasmin, calreticulin, annexin, type II regulatory subunit of protein kinase A (PKA) and rab GDP dissociation inhibitor and increases the cytosolic presence of adducin, actin, Ca2+-binding protein regucalcin/SMP30 and actin-depolymerizing factor.
    [Show full text]
  • Osteocalcin Improves Outcome After Acute Ischemic Stroke
    www.aging-us.com AGING 2020, Vol. 12, No. 1 Research Paper Osteocalcin improves outcome after acute ischemic stroke Jiayan Wu1, Yunxiao Dou1, Wangmi Liu2, Yanxin Zhao1, Xueyuan Liu1 1Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200032, China 2The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China Correspondence to: Xueyuan Liu, Yanxin Zhao, Wangmi Liu; email: [email protected], [email protected], [email protected] Keywords: osteocalcin, acute ischemic stroke, NIHSS score, proline hydroxylase 1, pyroptosis Received: November 6, 2019 Accepted: December 18, 2019 Published: January 5, 2020 Copyright: Wu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Background: Osteocalcin is related to energy metabolism, memory and the acute stress response, suggesting a relationship between bone and the brain. The need to explore the effect of osteocalcin on acute ischemic stroke is therefore urgent. Results: Patients with better outcomes had higher serum osteocalcin levels than those whose NIHSS scores did not improve. Multivariable logistic regression analysis showed acceptable performance (area under the curve = 0.766). The effect of osteocalcin on the promotion of neuron survival was confirmed by Cell Counting Kit-8 experiments. In addition, osteocalcin could decrease proline hydroxylase 1 and inhibit the degradation of gasdermin D. Conclusions: We propose that osteocalcin can improve outcome after acute ischemic stroke in the acute period. By downregulating proline hydroxylase 1, osteocalcin leads glucose metabolism to the pentose phosphate pathway and therefore promotes neuronal survival through inhibiting pyroptosis.
    [Show full text]
  • Calcium-Binding Proteins Annexin A2 and S100A6 Are Sensors of Tubular Injury and Recovery in Acute Renal Failure
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Kidney International, Vol. 68 (2005), pp. 2694–2703 Calcium-binding proteins annexin A2 and S100A6 are sensors of tubular injury and recovery in acute renal failure CHAO-WEN CHENG,ABDALLA RIFAI,SHUK-MAN KA,HAO-AI SHUI,YUH-FENG LIN,WEI-HWA LEE, and ANN CHEN Graduate Institute of Life Sciences, Graduate Institute of Medical Sciences, Department of Internal Medicine, Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taiwan, Republic of China; and Department of Pathology, Rhode Island Hospital, Rhode Island Calcium-binding proteins annexin A2 and S100A6 are sensors Acute tubular necrosis is the most common pathologic of tubular injury and recovery in acute renal failure. entity responsible for the clinical state of acute renal fail- Background. Rise in cellular calcium is associated with acute ure (ARF) [1, 2]. The two main causes of acute tubu- tubular necrosis, the most common cause of acute renal failure (ARF). The mechanisms that calcium signaling induce in the lar necrosis are ischemic and toxic injuries [3]. In the quiescent tubular cells to proliferate and differentiate during latter type, a variety of renal environmental substances acute tubular necrosis have not been elucidated. that include heavy metals such as mercury, lead, and ura- Methods. Acute tubular necrosis induced in mice by sin- nium are known to cause ARF.Nephrotoxic acute tubular gle intravenous injection of uranyl nitrate and examined af- necrosis is histologically evident as epithelial cell necrosis, ter 1, 3, 7, and 14 days.
    [Show full text]
  • Cell Surface-Expressed Phosphatidylserine As Therapeutic Target to Enhance Phagocytosis of Apoptotic Cells
    Cell Death and Differentiation (2013) 20, 49–56 & 2013 Macmillan Publishers Limited All rights reserved 1350-9047/13 www.nature.com/cdd Cell surface-expressed phosphatidylserine as therapeutic target to enhance phagocytosis of apoptotic cells K Schutters1, DHM Kusters1, MLL Chatrou1, T Montero-Melendez2, M Donners3, NM Deckers1, DV Krysko4,5, P Vandenabeele4,5, M Perretti2, LJ Schurgers1 and CPM Reutelingsperger*,1 Impaired efferocytosis has been shown to be associated with, and even to contribute to progression of, chronic inflammatory diseases such as atherosclerosis. Enhancing efferocytosis has been proposed as strategy to treat diseases involving inflammation. Here we present the strategy to increase ‘eat me’ signals on the surface of apoptotic cells by targeting cell surface- expressed phosphatidylserine (PS) with a variant of annexin A5 (Arg-Gly-Asp–annexin A5, RGD–anxA5) that has gained the function to interact with avb3 receptors of the phagocyte. We describe design and characterization of RGD–anxA5 and show that introduction of RGD transforms anxA5 from an inhibitor into a stimulator of efferocytosis. RGD–anxA5 enhances engulfment of apoptotic cells by phorbol-12-myristate-13-acetate-stimulated THP-1 (human acute monocytic leukemia cell line) cells in vitro and resident peritoneal mouse macrophages in vivo. In addition, RGD–anxA5 augments secretion of interleukin-10 during efferocytosis in vivo, thereby possibly adding to an anti-inflammatory environment. We conclude that targeting cell surface- expressed PS is an attractive strategy
    [Show full text]
  • Expression of Vascular Endothelial Growth Factor in Cultured Human Dental Follicle Cells and Its Biological Roles1
    Acta Pharmacol Sin 2007 Jul; 28 (7): 985–993 Full-length article Expression of vascular endothelial growth factor in cultured human dental follicle cells and its biological roles1 Xue-peng CHEN2, Hong QIAN2, Jun-jie WU2, Xian-wei MA3, Ze-xu GU2, Hai-yan SUN4, Yin-zhong DUAN2,5, Zuo-lin JIN2,5 2Department of Orthodontics, Qindu Stomatological College, Fourth Military Medical University, Xi’an 710032, China; 3Institute of Immunology, Second Military Medical University, Shanghai 200433, China; 4Number 307 Hospital of Chinese People’s Liberation Army, Beijing 100101, China Key words Abstract vascular endothelial growth factor; human Aim: To investigate the expression of vascular endothelial growth factor (VEGF) dental follicle cell; proliferation; differentia- in cultured human dental follicle cells (HDFC), and to examine the roles of VEGF in tion; apoptosis; tooth eruption; mitogen- activated protein kinase the proliferation, differentiation, and apoptosis of HDFC in vitro. Methods: Immunocytochemistry, ELISA, and RT-PCR were used to detect the expression 1 and transcription of VEGF in cultured HDFC. The dose-dependent and the time- Project supported by the National Natural Science Foundation of China (No 30400510). course effect of VEGF on cell proliferation and alkaline phosphatase (ALP) activ- 5 Correspondence to Dr Zuo-lin JIN and Prof ity in cultured HDFC were determined by MTT assay and colorimetric ALP assay, Yin-zhong DUAN. respectively. The effect of specific mitogen-activated protein kinase (MAPK) Phn 86-29-8477-6136. Fax 86-29-8322-3047. inhibitors (PD98059 and U0126) on the VEGF-mediated HDFC proliferation was E-mail [email protected] (Zuo-lin JIN) also determined by MTT assay.
    [Show full text]
  • Proteomic Analysis of Exosome-Like Vesicles Derived from Breast Cancer Cells
    ANTICANCER RESEARCH 32: 847-860 (2012) Proteomic Analysis of Exosome-like Vesicles Derived from Breast Cancer Cells GEMMA PALAZZOLO1, NADIA NINFA ALBANESE2,3, GIANLUCA DI CARA3, DANIEL GYGAX4, MARIA LETIZIA VITTORELLI3 and IDA PUCCI-MINAFRA3 1Institute for Biomedical Engineering, Laboratory of Biosensors and Bioelectronics, ETH Zurich, Switzerland; 2Department of Physics, University of Palermo, Palermo, Italy; 3Centro di Oncobiologia Sperimentale (C.OB.S.), Oncology Department La Maddalena, Palermo, Italy; 4Institute of Chemistry and Bioanalytics, University of Applied Sciences Northwestern Switzerland FHNW, Muttenz, Switzerland Abstract. Background/Aim: The phenomenon of membrane that vesicle production allows neoplastic cells to exert different vesicle-release by neoplastic cells is a growing field of interest effects, according to the possible acceptor targets. For instance, in cancer research, due to their potential role in carrying a vesicles could potentiate the malignant properties of adjacent large array of tumor antigens when secreted into the neoplastic cells or activate non-tumoral cells. Moreover, vesicles extracellular medium. In particular, experimental evidence show could convey signals to immune cells and surrounding stroma that at least some of the tumor markers detected in the blood cells. The present study may significantly contribute to the circulation of mammary carcinoma patients are carried by knowledge of the vesiculation phenomenon, which is a critical membrane-bound vesicles. Thus, biomarker research in breast device for trans cellular communication in cancer. cancer can gain great benefits from vesicle characterization. Materials and Methods: Conditioned medium was collected The phenomenon of membrane release in the extracellular from serum starved MDA-MB-231 sub-confluent cell cultures medium has long been known and was firstly described by and exosome-like vesicles (ELVs) were isolated by Paul H.
    [Show full text]
  • Annexin A2 Complexes with S100 Proteins
    British Journal of DOI:10.1111/bph.12978 www.brjpharmacol.org BJP Pharmacology Themed Section: Annexins VII Programme Correspondence Dr Lodewijk V Dekker, School of Pharmacy, Centre for REVIEW Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK. E-mail: Annexin A2 complexes [email protected] ---------------------------------------------------------------- Received with S100 proteins: 18 July 2014 Revised 16 September 2014 structure, function and Accepted 5 October 2014 pharmacological manipulation Yidong Liu, Helene K Myrvang and Lodewijk V Dekker School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, UK Annexin A2 (AnxA2) was originally identified as a substrate of the pp60v-src oncoprotein in transformed chicken embryonic fibroblasts. It is an abundant protein that associates with biological membranes as well as the actin cytoskeleton, and has been implicated in intracellular vesicle fusion, the organization of membrane domains, lipid rafts and membrane-cytoskeleton contacts. In addition to an intracellular role, AnxA2 has been reported to participate in processes localized to the cell surface including extracellular protease regulation and cell-cell interactions. There are many reports showing that AnxA2 is differentially expressed between normal and malignant tissue and potentially involved in tumour progression. An important aspect of AnxA2 function relates to its interaction with small Ca2+-dependent adaptor proteins called S100 proteins, which is the topic of this review. The interaction between AnxA2 and S100A10 has been very well characterized historically; more recently, other S100 proteins have been shown to interact with AnxA2 as well. The biochemical evidence for the occurrence of these protein interactions will be discussed, as well as their function.
    [Show full text]
  • Structure – Function Studies of Annexin A2 & A5
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by University of Minnesota Digital Conservancy Structure – Function Studies of Annexin A2 & A5 Stephanie Schramel Swenson College of Science and Engineering University of Minnesota Duluth [email protected] Introduction: Within the cell, proteins exist to maintain the intracellular level of calcium ions (Ca2+), allowing calcium to act as a secondary messenger. Annexins are a group of these proteins located in muscle cells which bind calcium in order to bind to phospholipids in the plasma membrane. Although there are multiple forms of this protein found in humans, all contain a similar structural basis consisting of an amino-terminal head domain, a carboxyl terminal domain, and conserved structural repeats of around 70 residues. Both the calcium binding and membrane binding sites are located at the carboxyl domain. Annexins bind to negatively charged phospholipids at the cellular membranes, which differentiates them from other calcium binding proteins. Exocytosis/endocytosis, ion transport, and transport of vesicles are also processes orchestrated by these proteins.1 Specifically, annexin A2 (AnxA2) and A5 (AnxA5) are the proteins we studied. Figure 1 shows the 3-D structure of AnxA2 with calcium bound. The question we tried to understand is the mechanism for how AnxA2 and AnxA5 function at the membrane in terms of the redistribution of lipids and the effect on membrane permeability. In order to study the annexins, we grew E. coli cells that expressed our desired proteins. We then went through multiple week- long purification processes to obtain pure protein to be used for further studies.
    [Show full text]