DOCSLIB.ORG

Quantitative Analysis of Gene Expression in Human Articular Cartilage from Normal and Osteoarthritic Joints I

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Quantitative Analysis of Gene Expression in Human Articular Cartilage from Normal and Osteoarthritic Joints I View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Osteoarthritis and Cartilage (2001) 9, 112–118 © 2001 OsteoArthritis Research Society International 1063–4584/01/020112+07 $35.00/0 doi:10.1053/joca.2000.0366, available online at http://www.idealibrary.com on Quantitative analysis of gene expression in human articular cartilage from normal and osteoarthritic joints I. Martin, M. Jakob, D. Scha¨fer, W. Dick, G. Spagnoli and M. Heberer Department of Surgery, Research Division, University of Basel, Switzerland Summary Objective: To quantify the expression of genes encoding extracellular matrix (ECM) proteins in human cartilage from normal and osteoarthritic (OA) joints. Design: Human cartilage samples were classified as control (CTR) or OA according to clinical evaluation and assessed histologically and biochemically to confirm the diagnosis. mRNAs encoding collagen types I, II and X, aggrecan, versican, osteopontin and osteocalcin were quantified by real-time reverse transcription-PCR assays and normalized to a reference mRNA (GAPDH). Results: RNA from native cartilage could be reproducibly and efficiently amplified by real-time PCR only if isolated using purification membranes. Primers and fluorescent probes for real-time PCR, endowed with comparable (<6% difference from GAPDH) and high (>91%) amplification efficiencies, were designed and validated for the selected ECM genes. The expression of most genes under investigation displayed large variations and was not significantly different in CTR and OA cartilage. Only osteopontin mRNA levels were significantly higher in OA than CTR specimens. mRNA ratios of collagen type II to I and of aggrecan to versican, defined as indexes of chondrocyte differentiation, were less variable within each population than the single genes and markedly higher (27.0 and 7.6-fold, respectively) in CTR than OA cartilage, with high statistical significance (P=0.00013 and P=0.00007, respectively). Conclusions: Our results provide evidence that gene patterns related to chondrocyte differentiation discriminate between CTR and OA human cartilage with higher sensitivity than single ECM genes. The method described here has the potential to improve understanding of the progression of OA and could become a valuable diagnostic tool. © 2001 OsteoArthritis Research Society International Key words: Real-time PCR, Chondrocyte, Extracellular matrix, Differentiation. Introduction OA cartilage samples7, and that collagen type I was abundantly expressed by chondrocytes of deep zones of The unique mechanical properties of articular cartilage OA fibrocartilaginous tissue8. Also the expression of differ- depend on the composition and organization of its extra- ent types of proteoglycans, investigated using a competi- cellular matrix (ECM). It is generally agreed that the pro- tive PCR technique, was not always consistent when gressive functional failure of cartilage in osteoarthrosis different sets of samples were used9,10. (OA) is not only due to enhanced degradation of the main The discordance among the different studies could be tissue components (e.g. type II collagen and aggrecan), but also to discoordinate changes in the types and amounts of due not only to variability among different individuals, but newly synthesized ECM molecules1. Thus, investigation of also to limitations inherent to the techniques used. Northern OA chondrocyte anabolic activity by quantification of the blotting and in-situ hybridization methods are not sensi- expression of mRNAs encoding specific ECM molecules tive enough to detect low-level gene expression and not should help understand the progression of the pathology, accurate enough to quantify the full range of expression. An and may ultimately improve its diagnosis and treatment. amplification step may therefore be required to quantify Despite the wealth of knowledge on the structure of mRNA amounts from limited tissue biopsies. However, different cartilage ECM molecules and their genes, there is quantification of mRNA using conventional PCR techniques relatively little and sometimes contradictory information on may be of limited accuracy, both because of the detection the quantitation of their expression in normal and OA method used (i.e. semi-quantitative image analysis), and articular cartilage. It was reported that mRNAs for collagen because analysis is often performed using a constant types I and X are absent in normal adult cartilage and that number of amplification cycles, after which the system collagen types II and X, but not I, are consistently could be in a saturation phase. up-regulated in specific layers of OA tissue2–6. However, Recently, real-time PCR has enabled the quantification independent studies performed using the same technique of mRNA with high accuracy, reproducibility and sensitivity in a wide dynamic range, without the need of post-PCR (i.e. in situ hybridization) reported that collagen type II 11 mRNA was detectable in only about 50% of the processed processing . In this paper, we developed and vali- dated real-time PCR assays to quantify the expression of genes encoding the most abundant molecules in the ECM Received 28 January 2000; accepted 16 May 2000 Address correspondence to: Ivan Martin, Research Division of of normal articular cartilage (i.e. collagen type II and the Department of Surgery, University of Basel, Hebelstrasse 20, aggrecan) and of degenerated fibrocartilage (i.e. collagen ZLF, Room 405, 4031 Basel, Switzerland. Tel.: + 41 61 265 2384; type I and versican), as well as of typical markers Fax: + 41 61 265 3990; E-mail: [email protected] of chondrocyte hypertrophy (i.e. collagen type X and 112 Osteoarthritis and Cartilage Vol. 9 No. 2 113 osteopontin) and of bone cells (i.e. osteocalcin). Our (1 mg/ml) containing 50 mM Tris, 1 mM EDTA, 1 mM iodo- hypothesis was that real-time PCR can lead to the identifi- acetamide, and 10 g/ml pepstatin A at 56°C for 15 h. cation of quantitative patterns of ECM gene expression, Proteinase K was then inactivated by boiling the digest for typical for normal and OA adult human cartilage tissue. 12 min. The GAG content was determined spectrophoto- metrically using dimethylmethylene blue dye and bovine chondroitin sulfate as a standard15. Collagen type II was Methods quantified using a monoclonal antibody-based inhibition ELISA14. PATIENTS AND SAMPLES Patient’s characteristics REAL-TIME QUANTITATIVE PCR Human articular OA cartilage was obtained from femoral heads, femoral condyles, and tibial plateaus of patients Theoretical basis operated for knee, hip or ankle replacement (10 individuals; Real-time quantitative PCR monitors the degradation of mean age 68.4, range 55–87) in the University Hospital of a sequence-specific, dual-labeled fluorescent probe after Basel. The diagnosis of OA was based on clinical and each cycle of PCR amplification. During the extension radiographic evaluations, according to standard criteria12 . phase, the 5′-exonuclease activity of Taq DNA polymerase Control (CTR) cartilage samples (seven individuals; mean cleaves the probe, separating the 5′-reporter fluorescent age 59.3, range 25–85) were collected from the hip or ankle dye from the 3′-quencher fluorescent dye, resulting in an of patients with no history of joint disease, undergoing increase in the emission spectra of the reporter fluorescent joint replacement following femoral neck fracture or foot dye. After subtraction of the background fluorescence, amputation for tumor resection. calculated during the first 15 amplification cycles, the measured fluorescence is graphed as an amplification plot. Cartilage specimens Each reaction is characterized by a value, Ct, defined as the fractional number of cycles at which the reporter Non-calcified cartilage (i.e. superficial, middle, and deep fluorescent emission reaches a fixed threshold level in the layers above the tidemark)3. was dissected from the bone exponential region of the amplification plot. The Ct value is immediately after surgery. Pieces of cartilage were correlated to input target mRNA amount: a larger starting obtained from the weight-bearing area of the joints, which quantity of mRNA target results in a lower number of PCR was macroscopically degenerated in OA patients, and cycles required for the reporter fluorescent emission to processed for the different assays as follows. Samples reach the threshold, and therefore a lower Ct value. Thus, were fixed in 4% buffered formalin at 4°C for 24 h for the method is not based on the measurement of the total histological analysis, frozen in pre-weighed vials for amount of amplified product after a fixed number of cycles, biochemical assessment, or homogenized in Trizol (Life as in conventional PCR, and does not require post-PCR Technologies, Basel, Switzerland) for mRNA quantitation. processing of the product11. Some samples were also fixed in acetone at 4°C for 10 min, or digested with 0.15% collagenase for 22 h to isolate primary chondrocytes. Primers and probes Primers and probes for human GAPDH, collagen types I, Osteoblast isolation II, and X, aggrecan, versican, osteopontin and osteocalcin were designed with the assistance of the Primer Express Bone chips from the iliac crest of patients undergoing computer program (Perkin-Elmer Applied Biosystems, spinal fusion were cultured in Petri dishes in medium Foster City, CA), in order to display minimal internal struc- supplemented with 10% fetal bovine serum, ascorbic acid, ture (i.e. primer–dimer formation) and similar melting tem- dexamethasone
Load more

Recommended publications
  • Versican V2 Assembles the Extracellular Matrix Surrounding the Nodes of Ranvier in the CNS
    The Journal of Neuroscience, June 17, 2009 • 29(24):7731–7742 • 7731 Cellular/Molecular Versican V2 Assembles the Extracellular Matrix Surrounding the Nodes of Ranvier in the CNS María T. Dours-Zimmermann,1 Konrad Maurer,2 Uwe Rauch,3 Wilhelm Stoffel,4 Reinhard Fa¨ssler,5 and Dieter R. Zimmermann1 Institutes of 1Surgical Pathology and 2Anesthesiology, University Hospital Zurich, CH-8091 Zurich, Switzerland, 3Vascular Wall Biology, Department of Experimental Medical Science, University of Lund, S-221 00 Lund, Sweden, 4Center for Biochemistry, Medical Faculty, University of Cologne, D-50931 Cologne, Germany, and 5Department of Molecular Medicine, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany The CNS-restricted versican splice-variant V2 is a large chondroitin sulfate proteoglycan incorporated in the extracellular matrix sur- rounding myelinated fibers and particularly accumulating at nodes of Ranvier. In vitro, it is a potent inhibitor of axonal growth and therefore considered to participate in the reduction of structural plasticity connected to myelination. To study the role of versican V2 during postnatal development, we designed a novel isoform-specific gene inactivation approach circumventing early embryonic lethality of the complete knock-out and preventing compensation by the remaining versican splice variants. These mice are viable and fertile; however, they display major molecular alterations at the nodes of Ranvier. While the clustering of nodal sodium channels and paranodal structures appear in versican V2-deficient mice unaffected, the formation of the extracellular matrix surrounding the nodes is largely impaired. The conjoint loss of tenascin-R and phosphacan from the perinodal matrix provide strong evidence that versican V2, possibly controlled by a nodal receptor, organizes the extracellular matrix assembly in vivo.
    [Show full text]
  • Supplementary Table 1: Adhesion Genes Data Set
    Supplementary Table 1: Adhesion genes data set PROBE Entrez Gene ID Celera Gene ID Gene_Symbol Gene_Name 160832 1 hCG201364.3 A1BG alpha-1-B glycoprotein 223658 1 hCG201364.3 A1BG alpha-1-B glycoprotein 212988 102 hCG40040.3 ADAM10 ADAM metallopeptidase domain 10 133411 4185 hCG28232.2 ADAM11 ADAM metallopeptidase domain 11 110695 8038 hCG40937.4 ADAM12 ADAM metallopeptidase domain 12 (meltrin alpha) 195222 8038 hCG40937.4 ADAM12 ADAM metallopeptidase domain 12 (meltrin alpha) 165344 8751 hCG20021.3 ADAM15 ADAM metallopeptidase domain 15 (metargidin) 189065 6868 null ADAM17 ADAM metallopeptidase domain 17 (tumor necrosis factor, alpha, converting enzyme) 108119 8728 hCG15398.4 ADAM19 ADAM metallopeptidase domain 19 (meltrin beta) 117763 8748 hCG20675.3 ADAM20 ADAM metallopeptidase domain 20 126448 8747 hCG1785634.2 ADAM21 ADAM metallopeptidase domain 21 208981 8747 hCG1785634.2|hCG2042897 ADAM21 ADAM metallopeptidase domain 21 180903 53616 hCG17212.4 ADAM22 ADAM metallopeptidase domain 22 177272 8745 hCG1811623.1 ADAM23 ADAM metallopeptidase domain 23 102384 10863 hCG1818505.1 ADAM28 ADAM metallopeptidase domain 28 119968 11086 hCG1786734.2 ADAM29 ADAM metallopeptidase domain 29 205542 11085 hCG1997196.1 ADAM30 ADAM metallopeptidase domain 30 148417 80332 hCG39255.4 ADAM33 ADAM metallopeptidase domain 33 140492 8756 hCG1789002.2 ADAM7 ADAM metallopeptidase domain 7 122603 101 hCG1816947.1 ADAM8 ADAM metallopeptidase domain 8 183965 8754 hCG1996391 ADAM9 ADAM metallopeptidase domain 9 (meltrin gamma) 129974 27299 hCG15447.3 ADAMDEC1 ADAM-like,
    [Show full text]
  • Role of Versican, Hyaluronan and CD44 in Ovarian Cancer Metastasis
    Int. J. Mol. Sci. 2011, 12, 1009-1029; doi:10.3390/ijms12021009 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Review Role of Versican, Hyaluronan and CD44 in Ovarian Cancer Metastasis Miranda P. Ween 1,2, Martin K. Oehler 1,3 and Carmela Ricciardelli 1,* 1 Research Centre for Reproductive Health, School of Paediatrics and Reproductive Health, Robinson Institute, University of Adelaide, Adelaide, South Australia 5005, Australia; E-Mails: [email protected] (M.P.W.); [email protected] (M.K.O.) 2 Research Centre for Infectious Diseases, School of Molecular Biosciences, University of Adelaide, South Australia 5005, Australia 3 Department of Gynaecological Oncology, Royal Adelaide Hospital, Adelaide, South Australia 5000, Australia * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +61-8-83038255; Fax: +61-8-83034099. Received: 30 November 2010; in revised form: 28 January 2011 / Accepted: 29 January 2011 / Published: 31 January 2011 Abstract: There is increasing evidence to suggest that extracellular matrix (ECM) components play an active role in tumor progression and are an important determinant for the growth and progression of solid tumors. Tumor cells interfere with the normal programming of ECM biosynthesis and can extensively modify the structure and composition of the matrix. In ovarian cancer alterations in the extracellular environment are critical for tumor initiation and progression and intra-peritoneal dissemination. ECM molecules including versican and hyaluronan (HA) which interacts with the HA receptor, CD44, have been shown to play critical roles in ovarian cancer metastasis. This review focuses on versican, HA, and CD44 and their potential as therapeutic targets for ovarian cancer.
    [Show full text]
  • Inflammation Amplification by Versican: the First Mediator
    Int. J. Mol. Sci. 2012, 13, 6873-6882; doi:10.3390/ijms13066873 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Opinion Inflammation Amplification by Versican: The First Mediator Zhenwei Zhang 1,†, Lei Miao 2,† and Lianghua Wang 1,* 1 Department of Biochemistry and Molecular Biology, Second Military Medical University, Shanghai 200433, China; E-Mail: [email protected] 2 Department of Pharmacology, Zhejiang Chinese Medical University, Hangzhou 310053, China; E-Mail: [email protected] † These authors contributed equally to this work. * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +86-21-81870970 (ext. 8011); Fax: +86-21-65334333. Received: 20 April 2012; in revised form: 3 May 2012 / Accepted: 29 May 2012 / Published: 6 June 2012 Abstract: The effects of inflammation may not always benefit the individual. Its amplifying nature represents a highly regulated biological program, and the inflammatory microenvironment is its essential component. Growing evidence suggests that the ECM (extracellular matrix) is important for the early steps of inflammation. Versican, a ubiquitous component of the ECM, contributes to the formation of the inflammatory response and is highly regulated by cytokines. Certain cytokines exert their initial effects on versican to alter the homeostasis of the inflammatory milieu, and inappropriate production of versican may promote the next inflammatory response. Therefore, versican could be the first step in the amplification of the inflammatory response, and ongoing research of this molecule may help to explain the pathogenesis of inflammation. Keywords: versican; cytokines; extracellular matrix homeostasis; inflammation amplification 1. Introduction Although inflammation helps to fight infection, it often results in an exacerbation of a diseased state in the individual [1].
    [Show full text]
  • Versican Upregulation in SÉZary Cells Alters
    Leukemia (2015) 29, 2024–2032 © 2015 Macmillan Publishers Limited All rights reserved 0887-6924/15 www.nature.com/leu ORIGINAL ARTICLE Versican upregulation in Sézary cells alters growth, motility and resistance to chemotherapy K Fujii1,3, MB Karpova1,4, K Asagoe1,5, O Georgiev2, R Dummer1 and M Urosevic-Maiwald1 Sézary syndrome (SéS) represents a leukemic variant of cutaneous T-cell lymphoma, whose etiology is still unknown. To identify dyregulated genes in SéS, we performed transcriptional profiling of Sézary cells (SCs) obtained from peripheral blood of patients with SéS. We identified versican as the highest upregulated gene in SCs. VCAN is an extracellular matrix proteoglycan, which is known to interfere with different cellular processes in cancer. Versican isoform V1 was the most commonly upregulated isoform in SCs. Using a lentiviral plasmid, we overexpressed versican V1 isoform in lymphoid cell lines, which altered their growth behavior by promoting formation of smaller cell clusters and by increasing their migratory capacity towards stromal cell-derived factor 1, thus promoting skin homing. Versican V1 overexpression exerted an inhibitory effect on cell proliferation, partially by promoting activation-induced cell death. Furthermore, V1 overexpression in lymphoid cell lines increased their sensitivity to doxorubicin and gemcitabine. In conclusion, we confirm versican as one of the dysregulated genes in SéS and describe its effects on the biology of SCs. Although versican overexpression confers lymphoid cells with increased migratory capacity, it also makes them more sensitive to activation-induced cell death and some chemotherapeutics, which could be exploited further for therapeutic purposes. Leukemia (2015) 29, 2024–2032; doi:10.1038/leu.2015.103 INTRODUCTION In this study, we identified versican as one of the highest Sézary syndrome (SéS), a leukemic variant of cutaneous T-cell upregulated genes in SCs using high-throughput gene expression lymphoma (CTCL), is characterized by erythroderma, generalized profiling.
    [Show full text]
  • Versican-Thrombospondin-1 Binding in Vitro and Colocalization in Microfibrils Induced by Inflammation on Vascular Smooth Muscle Cells
    Research Article 4499 Versican-thrombospondin-1 binding in vitro and colocalization in microfibrils induced by inflammation on vascular smooth muscle cells Svetlana A. Kuznetsova1, Philip Issa1, Elizabeth M. Perruccio1, Bixi Zeng1, John M. Sipes1, Yvona Ward2, Nicholas T. Seyfried3, Helen L. Fielder3, Anthony J. Day3, Thomas N. Wight4 and David D. Roberts1,* 1Laboratory of Pathology and 2Cell and Cancer Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA 3MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK 4The Hope Heart Program, Benaroya Research Institute at Virginia Mason, Seattle, WA 98101, USA *Author for correspondence (e-mail: [email protected]) Accepted 17 July 2006 Journal of Cell Science 119, 4499-4509 Published by The Company of Biologists 2006 doi:10.1242/jcs.03171 Summary We identified a specific interaction between two secreted poly-I:C-stimulated vascular smooth muscle cells, versican proteins, thrombospondin-1 and versican, that is induced organizes into fibrillar structures that contain elastin but during a toll-like receptor-3-dependent inflammatory are largely distinct from those formed by hyaluronan. response in vascular smooth muscle cells. Endogenous and exogenously added thrombospondin-1 Thrombospondin-1 binding to versican is modulated by incorporates into these structures. Binding of exogenous divalent cations. This interaction is mediated by interaction thrombospondin-1 to these structures, to purified versican of the G1 domain of versican with the N-module of and to its G1 domain is potently inhibited by heparin. thrombospondin-1 but only weakly with the corresponding At higher concentrations, exogenous thrombospondin-1 N-terminal region of thrombospondin-2.
    [Show full text]
  • Massive Aggrecan and Versican Accumulation in Thoracic Aortic Aneurysm and Dissection
    RESEARCH ARTICLE Massive aggrecan and versican accumulation in thoracic aortic aneurysm and dissection Frank S. Cikach,1 Christopher D. Koch,2,3 Timothy J. Mead,2 Josephine Galatioto,4 Belinda B. Willard,5 Kelly B. Emerton,6 Matthew J. Eagleton,7 Eugene H. Blackstone,8 Francesco Ramirez,4 Eric E. Roselli,8,9 and Suneel S. Apte2 1Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, USA. 2Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA. 3Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA. 4Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA. 5Proteomics and Metabolomics Core, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA. 6Cleveland Clinic Innovations, 7Department of Vascular Surgery, 8Department of Thoracic and Cardiovascular Surgery, and 9Aorta Center, Cleveland Clinic, Cleveland, Ohio, USA. Proteoglycan accumulation is a hallmark of medial degeneration in thoracic aortic aneurysm and dissection (TAAD). Here, we defined the aortic proteoglycanome using mass spectrometry, and based on the findings, investigated the large aggregating proteoglycans aggrecan and versican in human ascending TAAD and a mouse model of severe Marfan syndrome. The aortic proteoglycanome comprises 20 proteoglycans including aggrecan and versican. Antibodies against these proteoglycans intensely stained medial degeneration lesions in TAAD, contrasting with modest intralamellar staining in controls. Aggrecan, but not versican, was increased in longitudinal analysis of Fbn1mgR/mgR aortas. TAAD and Fbn1mgR/mgR aortas had increased aggrecan and versican mRNAs, and reduced expression of a key proteoglycanase gene, ADAMTS5, was seen in TAAD. Fbn1mgR/mgR mice with ascending aortic dissection and/or rupture had dramatically increased aggrecan staining compared with mice without these complications.
    [Show full text]
  • Syndecan - Regulation and Function of Its Glycosaminoglycan Chains
    Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 884 Syndecan - Regulation and Function of its Glycosaminoglycan Chains ANNA S. ERIKSSON ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-554-8637-2 UPPSALA urn:nbn:se:uu:diva-197691 2013 Dissertation presented at Uppsala University to be publicly examined in A1:107a, BMC, Husargatan 3, Uppsala, Friday, May 17, 2013 at 13:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Abstract Eriksson, A. S. 2013. Syndecan - Regulation and Function of its Glycosaminoglycan Chains. Acta Universitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 884. 54 pp. Uppsala. ISBN 978-91-554-8637-2. The cell surface is an active area where extracellular molecules meet their receptors and affect the cellular fate by inducing for example cell proliferation and adhesion. Syndecans and integrins are two transmembrane molecules that have been suggested to fine-tune these activities, possibly in cooperation. Syndecans are proteoglycans, i.e. proteins with specific types of carbohydrate chains attached. These chains are glycosaminoglycans and either heparan sulfate (HS) or chondroitin sulfate (CS). Syndecans are known to influence cell adhesion and signaling. Integrins in turn, are important adhesion molecules that connect the extracellular matrix with the cytoskeleton, and hence can regulate cell motility. In an attempt to study how the two types of glycosaminoglycans attached to syndecan-1 can interact with integrins, a cell based model system was used and functional motility assays were performed. The results showed that HS, but not CS, on the cell surface was capable of regulating integrin-mediated cell motility.
    [Show full text]
  • The Expression of Cell Surface Heparan Sulfate Proteoglycans and Their Roles in Turkey Skeletal Muscle Formation
    THE EXPRESSION OF CELL SURFACE HEPARAN SULFATE PROTEOGLYCANS AND THEIR ROLES IN TURKEY SKELETAL MUSCLE FORMATION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Xiaosong Liu, M.S. ***** The Ohio State University 2003 Dissertation Committee: Approved by Dr. Sandra G. Velleman, Advisor Dr. Karl E. Nestor Dr. Joy L. Pate _______________________ Advisor Dr. Wayne L. Bacon Department of Animal Sciences ABSTRACT Skeletal muscle myogenesis is a series of highly organized processes including cell migration, adhesion, proliferation, and differentiation that are precisely regulated by the extrinsic environment of muscle cells. Fibroblast growth factor 2 (FGF2) is one of the key growth factors involved in the regulation of skeletal muscle myogenesis. Since FGF2 is a potent stimulator of skeletal muscle cell proliferation but an intense inhibitor of cell differentiation, changes in FGF2 signaling to muscle cells will influence cell behavior and result in differences in cell proliferation and differentiation. As the cell surface heparan sulfate proteoglycans (HSPG), syndecans and glypicans, are the low- affinity receptors of FGF2 and function to regulate the binding of FGF2 to the high- affinity fibroblast growth factor receptors (FGFR) and affect the activity of FGF2, differences in the expression of these molecules may cause alterations in cell responsiveness to FGF2 stimulation, which can lead to changes in skeletal muscle development and growth. However, the precise functional differences of syndecans and glypicans in FGF2 signaling are unknown to date. Our hypothesis is that syndecans and glypicans may play different roles in regulating the FGF2-FGFR interaction, and the relative expression of these molecules is critical for determining the cell status in proliferation and differentiation.
    [Show full text]
  • Fibronectin Regulates Proteoglycan Production Balance in Transforming Growth Factor-Β1-Induced Chondrogenesis
    INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 28: 829-834, 2011 Fibronectin regulates proteoglycan production balance in transforming growth factor-β1-induced chondrogenesis TatsuHIKO KUTSUNA1,2, HIROFUMI INOUE2-4, HARUHIKO TAKEDA1, TOSHIAKI TAKAHASHI1, HARUYASU Yamamoto1, HIROMASA MIURA1 and SHIGEKI HIGASHIYAMA2,3 Departments of 1Bone and Joint Surgery and 2Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295; 3Department of Cell Growth and Tumor Regulation, 4Ehime-Nikon Bioimaging Core Laboratory, Ehime Proteo Medicine Research Center (ProMRes), Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan Received May 23, 2011; Accepted June 22, 2011 DOI: 10.3892/ijmm.2011.766 Abstract. Transforming growth factor (TGF)-β and bone Introduction morphogenetic protein (BMP) induce a cartilage-specific extracellular matrix (ECM) gene, aggrecan, in a chondrogenic Cartilage is a firm connective tissue with high water-holding cell line, ATDC5. The results of our recent study show that capacity found in many parts of animal bodies, including the TGF-β1, but not BMP-4, strongly induces an ECM gene, joints, ears, nose, trachea, and intervertebral discs. Cartilage fibronectin, during chondrogenesis. However, the role of fibro- is composed of chondrocytes which produce copious extra- nectin in chondrogenesis is unclear. In the current study, our cellular matrix (ECM), including type I or II collagen fibres, results showed that TGF-β1, but not BMP-4, led to versican- proteoglycans, and elastin fibres. Cartilage is classified into dominant proteoglycan production during chondrogenesis of 3 types, elastic cartilage, hyaline cartilage and fibrocartilage, ATDC5 cells. siRNA-mediated reduction of fibronectin and which differ in the relative amounts of the above-mentioned interference in the liaison between fibronectin and integrins main components.
    [Show full text]
  • Versican—A Critical Extracellular Matrix Regulator of Immunity and Inflammation
    REVIEW published: 24 March 2020 doi: 10.3389/fimmu.2020.00512 Versican—A Critical Extracellular Matrix Regulator of Immunity and Inflammation Thomas N. Wight 1*, Inkyung Kang 1, Stephen P. Evanko 1, Ingrid A. Harten 1, Mary Y. Chang 2, Oliver M. T. Pearce 3, Carys E. Allen 3 and Charles W. Frevert 2 1 Matrix Biology Program, Benaroya Research Institute at Virginia Mason, Seattle, WA, United States, 2 Division of Pulmonary/Critical Care Medicine, Center for Lung Biology, University of Washington School of Medicine, Seattle, WA, United States, 3 Centre for the Tumour Microenvironment, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom The extracellular matrix (ECM) proteoglycan, versican increases along with other ECM versican binding molecules such as hyaluronan, tumor necrosis factor stimulated gene-6 (TSG-6), and inter alpha trypsin inhibitor (IαI) during inflammation in a number of Edited by: different diseases such as cardiovascular and lung disease, autoimmune diseases, and Aaron C. Petrey, several different cancers. These interactions form stable scaffolds which can act as The University of Utah, United States “landing strips” for inflammatory cells as they invade tissue from the circulation. The Reviewed by: Anna Maria Piccinini, increase in versican is often coincident with the invasion of leukocytes early in the University of Nottingham, inflammatory process. Versican interacts with inflammatory cells either indirectly via United Kingdom hyaluronan or directly via receptors such as CD44, P-selectin glycoprotein ligand-1 Katherina Psarra, Evaggelismos General (PSGL-1), and toll-like receptors (TLRs) present on the surface of immune and Hospital, Greece non-immune cells.
    [Show full text]
  • The Role of Syndecan-1 in Cellular Signaling and Its Effects on Heparan
    REVIEW ARTICLE published: 19 December 2013 doi: 10.3389/fonc.2013.00310 The role of syndecan-1 in cellular signaling and its effects on heparan sulfate biosynthesis in mesenchymal tumors Tünde Szatmári and Katalin Dobra* Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden Edited by: Proteoglycans (PGs) and in particular the syndecans are involved in the differentiation Elvira V. Grigorieva, Institute of process across the epithelial-mesenchymal axis, principally through their ability to bind Molecular Biology and Biophysics SB growth factors and modulate their downstream signaling. Malignant tumors have indi- RAMS, Russia Reviewed by: vidual proteoglycan profiles, which are closely associated with their differentiation and Markus A. N. Hartl, University of biological behavior, mesenchymal tumors showing a different profile from that of epithelial Innsbruck, Austria tumors. Syndecan-1 is the main syndecan of epithelial malignancies, whereas in sarcomas Swapna Asuthkar, University of Illinois its expression level is generally low, in accordance with their mesenchymal phenotype and College of Medicine at Peoria, USA highly malignant behavior. This proteoglycan is often overexpressed in adenocarcinoma *Correspondence: Katalin Dobra, Department of cells, whereas mesothelioma and fibrosarcoma cells express syndecan-2 and syndecan-4 Laboratory Medicine, Karolinska more abundantly. Increased expression of syndecan-1 in mesenchymal tumors changes Institutet, Karolinska University the tumor cell morphology to an epithelioid direction whereas downregulation results in Hospital F-46, SE-141 86 Stockholm, a change in shape from polygonal to spindle-like morphology. Although syndecan-1 plays Sweden e-mail: [email protected] major roles on the cell-surface, there are also intracellular functions, which are not very well studied.
    [Show full text]