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T IMMERMANS, Department of Veterinary Sciences, University of Antwerpen, Groenenborgerlaan 171, 2020 Antwerpen, Belgium e-mail: [email protected] 197 Advances in Anatomy Embryology and Cell Biology Editors F.F. Beck, Melbourne . F. Clascá, Madrid M. Frotscher, Freiburg . D.E. Haines, Jackson N. Hirokawa, Tokyo . Z. Kmiec, Gdansk H.-W. Korf, Frankfurt . E. Marani, Enschede R. Putz, München . Y. Sano, Kyoto T.H. Schiebler, Würzburg J.-P. Timmermans, Antwerpen M. Shakibaei , C. Csaki and A. Mobasheri Diverse Roles of Integrin Receptors in Articular Cartilage With 36 Figures Mehdi Shakibaei Constanze Csaki Institute of Anatomy Musculoskeletal Research Group Ludwig-Maximilian-University Munich Pettenkoferstrasse 80336 Munich Germany e-mail: [email protected] Ali Mobasheri Division of Comparative Veterinary Medicine School of Veterinary Medicine and Science University of Nottingham Sutton Bonington Campus Loughborough Leicestershire LE12 5RD UK ISSN 0301-5556 ISBN 978-3-540-78770-9 e-ISBN 978-3-540-78771-6 Library of Congress Control Number: 2008923175 © 2008 Springer-Verlag Berlin Heidelberg This work is subject ot copyright. All rights are reserved, whether the whold or part of the m aterial is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, roadcasting reporduction on microfilm or in any other way, and storage in data banks. 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Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com List of Contents 1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1 Integrins: Multifunctional Adhesion Molecules . . . . . . . . . . . . . . . . . . . . 4 1.2 Integrin Structure–Function Relationships. . . . . . . . . . . . . . . . . . . . . . . . 6 1.3 Structure of Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.4 EGF Domains in β1-Integrins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2 Integrins in Articular Cartilage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.1 Monolayer Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.1.1 Expression Pattern and Changes of Integrins on Chondrocytes in Monolayer Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.1.2 Integrin Expression and Collagen Type II are Implicated in the Maintenance of Chondrocyte Morphology in Monolayer Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2.1.3 Signal Transduction by β1-Integrin Receptors in Chondrocytes In Vitro: Collaboration with IGF-IR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 2.1.4 Inhibition of MAPK Pathway Induces Chondrocyte Apoptosis . . . . . . . 19 2.1.5 Expression of the VEGF Receptor-3 in Osteoarthritic Chondrocytes and Association with β1-Integrins. . . . . . . . . . . . . . . . . . . 22 2.1.6 Effects of Curcumin on IL-1β-Induced Inhibition of Collagen Type II, β1-integrin Synthesis and Activation of Caspase-3 in Human Chondrocytes In Vitro. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.2 High-Density Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 2.2.1 ECM Changes Following Long-Term Cultivation of Cartilage (Organoid/High-Density Cultures). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 2.2.2 Expression of Integrins in Ageing Cartilage Tissue In Vitro . . . . . . . . . . 31 2.2.3 Changes in Integrin Expression During Chondrogenesis In Vitro. . . . . 32 2.2.4 Inhibition of Chondrogenesis by Incubating Chondrocyte Cultures with an Anti-integrin Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . 35 2.2.5 Integrin Expression During Differentiation of Mesenchymal Limb Bud Cells to Chondrocytes in Alginate Culture. . . . . . . . . . . . . . . . 36 2.2.6 β1-Integrins Exist in the Cartilage Matrix . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.2.7 Integrins and Matrix Metalloproteinases Co-localise in the Extracellular Matrix of Chondrocyte Cultures . . . . . . . . . . . . . . . . . . . . . 40 vi List of Contents 2.2.8 Integrins and Stretch-Activated Cation Channels: Putative Components of Chondrocyte Mechanoreceptors . . . . . . . . . . 42 2.2.9 β1-Integrins Co-localise with Selected Ion Channels in Mechanoreceptor Complexes of Mouse Limb-Bud Chondrocytes. . . . 44 2.2.10 Inhibition of Integrin Function Results in Chondrocyte Apoptosis. . . 46 3 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Subject Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Introduction 1 1 Introduction A rticular cartilage is a specialised connective tissue with unique biological and mechanical properties which depend on the structural design of the tissue and the interactions between its unique resident cells, the chondrocytes, and the extracel- lular matrix (ECM) that makes up the bulk of the tissue (Buckwalter and Mankin 1998). Chondrocytes (Fig. 1 ) are the architects of the ECM (Muir 1995), building the macromolecular framework of the ECM from three distinct classes of macro- molecules: collagens, proteoglycans, and noncollagenous proteins. Of the collagens present in articular cartilage, collagens type II, IX, and XI form a fibrillar meshwork that gives cartilage tensile stiffness and strength (Eyre 2004; Buckwalter and Mankin 1998; Kuettner et al. 1991), whereas collagen type VI forms part of the matrix imme- diately surrounding the chondrocytes, enabling them to attach to the macromo- lecular framework of the ECM and acting as a transducer of biomechanical and biochemical signals in the articular cartilage (Guilak et al. 2006; Roughley and Lee 1994). Embedded in the collagen mesh are large aggregating proteoglycans (aggre- can), which give cartilage its stiffness to compression, its resilience and contribute to its long-term durability (Dudhia 2005; Kiani et al. 2002; Luo et al. 2000; Roughley and Lee 1994). The extracellular matrix proteins in cartilage are of great significance for the regulation of the cell behaviour, proliferation, differentiation and morphogenesis (Kosher et al. 1973; Kosher and Church 1975; von der Mark et al. 1977; Hewitt et al. 1982; Sommarin et al. 1989; Ramachandrula et al. 1992; Ruoslahti and Reed 1994; Enomoto-Iwamoto et al. 1997; Gonzalez et al. 1993). Further, embedded in the meshwork are small proteoglycans, including d ecorin, biglycan and fibromodulin. Decorin and fibromodulin both interact with the type II collagen fibrils in the matrix and play a role in fibrillogenesis and interfibril interactions. Biglycan is mainly found in the immediate surrounding of the chondrocytes, where it may interact with collagen type VI (Buckwalter and Mankin 1998; Roughley and Lee 1994). Modulation of the ECM proteins is regulated by an interaction of a diversity of growth factors with the chondrocytes (Jenniskens et al. 2006; Trippel et al. 1989; Isgaard 1992; Hunziker et al. 1994; Sah et al. 1994). In fact, it has been reported recently that IGF-I and TGF-β stimulate the membrane expression of integrins, and that this event is accompanied by increasing adhesion of chondrocytes to matrix proteins (Loeser 1997). Other noncollagenous proteins in articular cartilage such as cartilage oligomeric matrix protein (COMP) are less well studied and may have value as a marker of cartilage turnover and degeneration (Di Cesare et al. 1996), while tenascin and fibronectin influence interactions between the chondrocytes and the ECM (Buckwalter and Mankin 1998; Burton-Wurster et al. 1997). The major cellular and molecular components of articular cartilage are shown in Fig. 2 . T he ECM surrounds chondrocytes and protects them from biomechanical stress arising during normal joint motion, determines the types and concentrations of mol- ecules that reach the cells and helps to maintain the chondrocyte p henotype. Throughout life, cartilage undergoes continuous internal remodeling as chondrocytes replace 2 Introduction M M C C M M C Fig. 1 Electron microscopic demonstration of chondrocytes. Typical chondrocyte ( C ) with smooth surface and numerous cavities of rough endoplasmic reticulum, mitochondria, other cell organelles, vacuoles and granules. Chondrocytes are embedded in a network of extra cellular matrix of thin irregularly running fibrils (M ) closely attached to the cell surface (a rrows ) A B COMP Chondrocyte Aggrecan Fibronectin Hyaluronan Collagen IX Decorin Collagen II Fibromodulin Biglycan Thrombospondin Fig. 2A, B Light microscopic appearance of equine articular cartilage and the major cellu- lar and molecular components of articular cartilage. A Histological morphology of normal equine articular cartilage showing a relatively homogeneous tissue structure consisting of a single cell type, the chondrocyte. B Chondrocytes synthesise and maintain a macromolecular framework consisting of three distinct classes of macromolecules: collagens, proteoglycans, and noncollagenous proteins. Collagens (type II, IX, and XI) form the fibrillar meshwork of the ECM, whereas collagen type VI forms part of the peri-cellular matrix. Embedded in the collagen mesh are large aggregating proteoglycans (aggrecan). Also embedded in the matrix are small proteoglycans, including decorin, biglycan, fibromodulin and other noncolla- genous proteins such as cartilage oligomeric matrix protein (COMP) and fibronectin matrix macromolecules lost through degradation. Evidence indicates that ECM turnover depends on the ability of chondrocytes to detect alterations in the mac- romolecular composition and organisation of the matrix, including the presence of Introduction 3 degraded macromolecules, and to respond by s ynthesising appropriate types and amounts of new ECM components. It is known that mechanical loading of cartilage creates mechanical, electrical, and physicochemical signals that help to direct the syn- thesising and degrading activity of chondrocytes (Fig. 3 ) (Mobasheri et al. 2002a). In addition, the ECM acts as a signal transducer for the chondrocytes (Millward-Sadler and Salter 2004). A prolonged and severe decrease in the joint loading and usage leads to alterations in the composition of the ECM and eventually to loss of tissue struc- ture and its specific biomechanical properties, whereas normal physical load on the joint stimulates the biosynthetic activity of chondrocytes and possibly the internal A B Resting Cartilage EExxttrraacceelllluullaarr MMaattrriixx Na+, K+ Ca2+, Cl− H2O ∆ψ CChhoonnddrrooccyyttee ∆π* Pressure = 1 atm [Na+] = 240-300 mM PPOO 350 mOsm Normal cell volume Load Loaded Cartilage PA Pressure = 50-200 atm [Na+] = 250-350 mM 380-480 mOsm Cell shrinkage leading to the elevation of local cation concentrations (Na+, K+ and Ca2+) and activation of volume regulatory ion and osmolyte transport systems Possible changes to the cell membrane potential and activity of ion channels. Fig. 3A, B The effects of mechanical load and the extracellular ionic and osmotic milieu on the physical environment of human articular chondrocytes. A Chondrocytes are excel- lent sensors of mechanical signals and respond to these signals in coordination with other environmental, hormonal and genetic factors to regulate metabolic activity. Resting articu- lar cartilage experiences normal atmospheric pressure equivalent to 1 atmosphere (a tm ). Mechanically loaded articular cartilage is exposed to pressures as high as 50–200 atm. Mechanical loading of cartilage results in cell shrinkage, which in turn leads to the elevation of local cation concentrations (Na + , K + and Ca 2+ ) and the activation of volume regulatory ion and osmolyte transport systems. In addition, there are changes to the cell membrane potential and activity of ion channels. B Chondrocytes are also excellent sensors of ionic and osmotic signals. This schematic illustrates the mechano-electrochemical responses in chondrocytes under mechanical load and the interaction between the extracellular matrix and chondrocytes. ∆π* represents changes in osmotic pressure and ∆ψ symbolises the elec- trical membrane potential difference across the chondrocyte plasma membrane. (Adapted from Mobasheri et al. 2002)