Cartilage oligomeric matrix protein and its binding partners in the cartilage extracellular matrix: Interaction, regulation and role in chondrogenesis

Matrix Biology 37 (2014) 102–111

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Matrix Biology journal homepage: www.elsevier.com/locate/matbio

Mini review

Cartilage oligomeric matrix protein and its binding partners in the cartilage extracellular matrix: Interaction, regulation and role in chondrogenesis Chitrangada Acharya a, Jasper H.N. Yik a, Ashleen Kishore a, Victoria Van Dinh a, Paul E. Di Cesare b, Dominik R. Haudenschild a,⁎ a b

Department of Orthopaedic Surgery, Lawrence J. Ellison Musculoskeletal Research Center, University of California at Davis Medical Center, Sacramento, CA 95817, USA Department of Orthopaedics and Rehabilitation, New York Hospital Queens, New York, NY 11355, USA

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Available online 2 July 2014 Keywords: Cartilage oligomeric matrix protein Extracellular matrix Receptors Proteases Growth factors Complement system

a b s t r a c t Thrombospondins (TSPs) are widely known as a family of five calcium-binding matricellular proteins. While these proteins belong to the same family, they are encoded by different genes, regulate different cellular functions and are localized to specific regions of the body. TSP-5 or Cartilage Oligomeric Matrix Protein (COMP) is the only TSP that has been associated with skeletal disorders in humans, including pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). The pentameric structure of COMP, the evidence that it interacts with multiple cellular proteins, and the recent reports of COMP acting as a ‘lattice’ to present growth factors to cells, inspired this review of COMP and its interacting partners. In our review, we have compiled the interactions of COMP with other proteins in the cartilage extracellular matrix and summarized their importance in maintaining the structural integrity of cartilage as well as in regulating cellular functions. © 2014 Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . Structure of COMP/TSP-5. . . . . . . . . . . . . . . . Interaction of COMP with extracellular matrix proteins . . 3.1. Interaction with collagens . . . . . . . . . . . . 3.2. Interaction with aggrecan and chondroitin sulfates. 3.3. Interaction with fibronectin . . . . . . . . . . . 3.4. Interaction with matrilin . . . . . . . . . . . . 4. Interaction of COMP with cell surface proteins . . . . . . 5. Interaction of COMP with extracellular proteases . . . . . 6. Interaction of COMP with hydrophobic compounds . . . . 7. Interaction of COMP with the complement system . . . . 8. Interaction of COMP with growth factors . . . . . . . . 9. Conclusions. . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

⁎ Corresponding author at: Department of Orthopaedic Surgery, Lawrence J. Ellison Musculoskeletal Research Center, University of California Davis Medical Center, Suite 2000, Sacramento, CA 95817, USA. Tel.: +1 916 734 5015; fax: +1 916 734 5750.

The thrombospondins (TSPs) are a family of five matricellular calcium-binding proteins that participate in cellular responses to growth factors, cytokines and injury (Chen et al., 2000). These proteins seem to function mainly as adapter molecules to guide extracellular matrix (ECM) synthesis and tissue remodeling in various physiological and

http://dx.doi.org/10.1016/j.matbio.2014.06.001 0945-053X/© 2014 Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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pathological conditions (Tan and Lawler, 2009). Group A TSPs (TSP1 and TSP2) are homotrimers, whereas group B TSPs (TSP3, − 4, and − 5/ COMP) are homopentamers (Adams, 2001), and all the TSPs are secreted as disulfide-bonded complexes. Thrombospondins play a significant role in the determination of cellular phenotype (Tan et al., 2009), and while they may be differentially expressed as a component of ECM in different organs, they are found in abundance only in specific organs of the body. For instance, TSP-1, the first to be discovered and the most widely studied TSP, is found in the epithelial layer of skin, blood and myocardium. TSP-2 is found in the skin and tendons, TSP-3 in lung, cartilage and brain tissues, TSP4 in the skeletal tissue, neural tissue and tendons of developing embryos and, while TSP-5 is mainly found in cartilage and bone tissue (Müller et al., 1998; Tan and Lawler, 2009), its presence has also been reported in other connective tissues like skin (Agarwal et al., 2012), vascular smooth muscle cells (Wang et al., 2010), tendons and ligaments (Müller et al., 1998). The major significance of TSPs in the ECM lies in the fact that ECM deficient in a particular TSP leads to cellular and physiological derangements within the living body. TSP-1 null mice exhibit a decrease in collagen content, delayed macrophage recruitment in wound closure, as well as delayed re-epithelialization of wounds (DiPietro et al., 1996; Agah et al., 2002). TSP-2 null mice have more elastic skin, flexible tails, increased bone and cortical bone density on the account of loosely packed and therefore less organized collagen fibrils (Kyriakides et al., 1998). TSP-3 null mice display a decrease in columnar arrangement of chondrocytes in the growth plate, and as a consequence a decrease in limb length (Posey et al., 2008). While TSP-4 has a major role in the neuromuscular junctions of developing chick embryos (Arber and Caroni, 1995), the pathophysiological function of this protein has not been well studied. A well-characterized functional genetic polymorphism of TSP-4 (A387P) has been associated with an increased coronary risk in post-infarction patients with high density lipoprotein C and C-reactive protein (Corsetti et al., 2011). TSP-5 is also known as cartilage oligomeric matrix protein (COMP) and is the only member of this family which has been linked to a skeletal disorder in humans. The gene for COMP was localized to 19p13.1 and mutations within this gene lead to pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) (Briggs et al., 1995; Hecht et al., 1995), which are characterized by severe to short limb dwarfism with normal skull development. These are autosomal dominant inherited forms of osteochondrodysplasias, and the major clinical complications caused by such conditions are premature osteoarthritis (OA) of load bearing joints, and abnormalities of the epiphyses of hands, long bones and hips (Cohn et al., 1996; Thur et al., 2001). COMP is remarkably conserved across mammalian species and shows differential expression at different stages of development. In studies associated with murine limb development, COMP was detected as early as day 10 of gestation in the condensing mesenchyme. At day 13, COMP is present in all cartilaginous tissues and skeletal muscles of mouse embryo, while at day 19, the distribution is restricted to hypertrophic zone of growth plate, perichondrium and periosteum, and the superficial zone of articular cartilage. Such restricted patterns of COMP distribution and expression in developing and adult murine tissues point toward the tightly controlled transcriptional regulation of COMP (Fang et al., 2000). COMP interacts with a number of cartilage ECM proteins, including fibronectin, collagen I, II IX, XII, and XIV, matrilins, as well as proteoglycans such as aggrecan and others (Thur et al., 2001; Briggs and Chapman, 2002; Di Cesare et al., 2002; Budde et al., 2005; Chen et al., 2007). Through these interactions, COMP plays an important role in matrix assembly. COMP can also have an active role in the immune system by triggering the alternate pathway of the complement system (Happonen et al., 2010, 2012b). There are reports suggesting autoimmunity to COMP in rats and mice, and evidence that COMP-specific antibodies are pathogenic (Carlsen et al., 2008; Geng et al., 2012; Kinne, 2013). Earlier studies revealed that increased levels of COMP are

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found in OA and rheumatoid arthritis (RA) and these increased levels in synovial fluid and serum are utilized as biomarkers for such pathological conditions (Di Cesare et al., 1996; Neidhart et al., 1997; Petersson et al., 1998). Taken together, these studies reflect the necessity for a deeper understanding of COMP binding to other proteins. COMP is a homopentamer, which gives the molecule additional flexibility to interact with a greater number of molecules and act as a bridging molecule between a protein of interest and its activating partner, or between the proteins and the cell surface. Such features open-up a variety of possibilities for new molecular interactions and cellular functions. Recent research has shown that COMP not only binds to different ECM components but it also interacts with growth factors and acts as a ‘lattice’ to present them for utilization by the cells (Haudenschild et al., 2011; Ishida et al., 2013). This intriguing activity of COMP suggests its possible involvement in influencing vital cell functions like cell attachment, proliferation and differentiation, by modulating the ECM and growth factor interaction with cells. In light of this new evidence, the purpose of this review is to focus on the binding interactions of COMP with the ECM proteins as well as growth factors, and the significance of these binding interactions in modulating cellular phenotype or function. 2. Structure of COMP/TSP-5 COMP is a secreted multidomain glycoprotein that forms a disulfidebonded homopentamer of ~524 kDa. In the earlier studies of cartilage matrix proteins, COMP was described as “an oligomeric protein of molecular weight more than 500 kDa with subunits of 100 kDa”, found in the developing mouse limbs and in cultures of limb bud mesenchyme. It was found to be distributed along with type II collagen throughout the cartilaginous nodules by day 14 of gestation, and by day 18 it was found in the distal and proximal part of the cartilage (Franzen et al., 1987). COMP was first isolated from bovine articular cartilage by extraction under denaturing conditions with 4M guanidine-HCl (Hedbom et al., 1992). The monomeric form comprises an amino-terminal coiled-coil domain, four type 2 epidermal growth factor (EGF)like domains, eight type 3 calcium binding calmodulin-like or thrombospondin-like domains and a globular carboxy-terminal domain (Newton et al., 1994). The N-terminal coiled-coil domain mediates pentamerization, resulting in a bouquet-like arrangement of the five monomers (Mörgelin et al., 1992; Malashkevich et al., 1996), and forming a cavity in which different hydrophobic compounds like Vitamin D3 and all-trans retinol can reside (Guo et al., 1998). COMP belongs to subgroup B of the TSP family, alongside TSP-3 and TSP-4, all of which comprise four EGF-like domains, as opposed to TSP-1 and TSP-2 that have three such domains (Kvansakul et al., 2004; Carlson et al., 2005). The EGF-like repeats, the type 3 repeats and the C-terminal domain fold to form a domain assembly, which is designated as a signature domain due to its occurrence in all TSP family members (Kvansakul et al., 2004; Carlson et al., 2005). Among the approximately 100 disease-causing COMP mutations reported, the majority occurs within the type 3 calcium binding domains and a few occur in the C-terminal domain (Carlson et al., 2005). These mutations are mainly single amino acid mutations, but also include insertions, deletions, and point mutations. Many of these naturally occurring COMP mutations can cause skeletal disorders in humans. For example, PSACH can be caused by an aspartate deletion at amino acid number 469 (D469Δ), a mutation that is discussed in more detail below, and also my many other point mutations in the coding sequence of COMP. Several other mutations in COMP can lead to MED, such as the substitution of aspartate at position 361 by tyrosine (D361Y). These mutations cause COMP to be retained in the large cisternae of the rough endoplasmic reticulum (rER), thus very little COMP is deposited in the matrix, leading to compromised chondrocyte function and cell death (Hecht et al., 1998; Hashimoto et al., 2003). Immunohistochemistry and quantitative RT-PCR results showed D469Δ-COMP (mutant

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COMP) causes chondrocyte apoptosis in the first postnatal week. Necroptosis was observed as a result of inflammation, oxidative stress, and DNA damage in 3 weeks, when most chondrocytes retain D469ΔCOMP. This was believed to be a result of an interaction between the mutant COMP and the chaperone protein CHOP (Ddit) (Posey et al., 2012). The quantity of matrix was reduced due to decreased chondrocyte numbers (Hecht et al., 1998; Hashimoto et al., 2003). Additionally, this impaired the secretion of other proteins like type IX collagen and matrilin-3, thereby affecting the matrix quality significantly (Hecht et al., 2005). PSACH and MED patients showed very little amounts of mutant COMP secreted into the matrix (the majority is retained in the rER), and this COMP had reduced binding to calcium as well as types I, II and IX collagens (Thur et al., 2001). Other studies suggest that cartilage matrix integrity is compromised by mutant COMP even when it is efficiently secreted (Schmitz et al., 2006; Hansen et al., 2011). There are up to 30 cation binding sites in a mature pentameric COMP molecule, mostly occupied by calcium (Tan et al., 2009). Divalent cations like zinc, calcium, magnesium, manganese, and their chelation by agents such as EDTA, affect the binding of COMP to other ECM proteins. In severe PSACH and MED, the concentration for half-maximal saturation of COMP binding to types I, II and IX collagens was lowered in the presence of Zn, leading to better binding (Thur et al., 2001). The crystal structure of a COMP mutant comprising the last of the EGF-like domain, the entire type 3 domain and the C-terminal domain revealed the presence of a metal ion dependent adhesion site (MIDAS), which enables interaction with collagens, and various points of interaction of the type 3 repeats with the globular C-terminal domain (Tan et al., 2009). Thus, cations are important contributors to the different conformations of COMP and cations influence the interaction of COMP with other components of the extracellular matrix. In summary, COMP is a secreted pentameric multi-domain glycoprotein of the thrombospondin family. It forms an integral part of the cartilage ECM, and mutant COMP (seen in PSACH and MED) shows not only reduced COMP, type IX collagen and matrilin-3 deposition but also reduced overall matrix formation and increased chondrocyte death. It therefore becomes imperative to study the interactions of COMP with other proteins, and the effects of these interactions on tissue and cellular functions. Given the modular structural components of COMP, many possibilities exist for binding interactions. This review focuses on those interactions supported by direct observations in the literature. 3. Interaction of COMP with extracellular matrix proteins COMP is abundantly expressed in articular cartilage as well as in proliferative and hypertrophic chondrocytes of the growth plate, implicating its importance in endochondral ossification and development of articular cartilage (DiCesare et al., 1995; Rock et al., 2010). COMP was first known to interact with ECM proteins and acts as a bridge between these molecules on the account of its multi-domain modular structure. Following is an account of its interactions with various ECM constituents. 3.1. Interaction with collagens Collagens comprise a large family of molecules with 28 different members. In cartilage and tendons, type II and type I collagens are the major collagen components, respectively providing the tissues with resistance to tensile forces. Collagens typically have three α chains forming a stable triple helical structure. COMP binds significantly to type I and type II collagens as well as procollagens I and II in the presence of Zn2+, at four defined sites on the collagen and procollagen molecules: two located close to either end, and the other two located 126 and 206 nm from the C-terminus. The dissociation constants for COMP binding to collagens were 1.50 ± 0.25 nM and 1.72 ± 0.16 nM for type I and type II collagens, respectively. The COMP–collagen interaction is probably mediated by one class of binding site present at the C-terminal globular domain of

COMP (Rosenberg et al., 1998). In its pentameric state, COMP is also known to promote fibrillogenesis of type I and type II collagens. COMP interacts mainly with free type I and type II collagen molecules, bringing several of these molecules closer, promoting further assembly (Halász et al., 2007). The binding of COMP to type IX collagen, which is located at the surface of type II collagen fibrils, indicates that COMP acts as a “bridging molecule” involved in matrix assembly, thereby enhancing the stability of the fibrillar network. The mutant COMP molecules associated with PSACH and MED syndromes exhibit reduced binding to type IX collagen, and this might lead to a weak or less stable fibrillar networks associated with these diseases (Thur et al., 2001). It may be significant to note that the single amino acid mutations leading to severe PSACH and MED are present in the calcium-binding type 3 domain while the binding site of types I-, II- and IX-collagens remains in the C-terminal domain of COMP. Among early events of cartilage degradation, there is a fragmentation of type IX collagen and loss of its NC4 domain, which normally projects out from the fibril surface and provides binding sites for COMP. This NC4 domain also functions as an inhibitor of the complement system and binds to C4, C3 and C9 components and directly inhibits C9 polymerization and assembly of the lytic membrane attack protein. The NC4 domain of type IX collagen and its interaction with COMP may thus protect the cartilage from complement activation and chronic inflammation as seen in diseases like rheumatoid arthritis (Kalchishkova et al., 2011). Immunolocalization of various matrix proteins in PSACH cartilage revealed that several components were retained in the rough endoplasmic reticulum (rER) along with mutant COMP, while others were secreted normally. Types II and VI collagens were secreted normally while type IX collagen was retained at high levels in rER cisternae (Maddox et al., 1997; Délot et al., 1998; Vranka et al., 2001; Hecht et al., 2005). Types XI and XII collagens were present in the rER cisternae at much lower levels, indicating different secretory pathways or sorting mechanisms for different matrix components. This retention increased local concentrations of mutant COMP and may facilitate interactions with other matrix molecules, resulting in the formation of organized protein aggregates within the rER (Vertel et al., 1989; Vranka et al., 2001). Recently a binding interaction between COMP and collagen types XII and XIV was described in the dermal compartment of healthy skin. This interaction occurs via the C-terminal collagenous domains, and in this context COMP may also function as an adaptor to organize anchoring plaques and the dermal collagen network (Agarwal et al., 2012). The above examples indicate that COMP–collagen interactions are extremely vital to the maintenance of the cartilage ECM. Any degradation or mutation associated with either collagen or COMP, or both, affects the secretion of these proteins as well as the overall fibrillar organization of the matrix. 3.2. Interaction with aggrecan and chondroitin sulfates The pericellular matrix secreted by chondrocytes mainly comprises of glycosaminoglycans (GAGs) and GAG-containing proteoglycans. One of the major components of this class of protein in the matrix is aggrecan. The presence of aggrecan aggregates, in the meshwork of collagen fibrils, confers to the articular cartilage the ability to resist compression. The high density of negative charges of the GAG chains draws water into the tissue, thereby increasing the osmotic pressure. The balance between the osmotic pressure of the proteoglycan and the tension in the collagen fibers contributes to the ability of articular cartilage to bear compressive loads (Chandran and Horkay, 2012). COMP binds to aggrecan in a concentration-dependent manner (Chen et al., 2007). When calcium was chelated from the full-length COMP using EDTA, the binding was diminished drastically. It was found that a deletion mutation (D469) in the calcium-binding domain caused COMP to bind with decreased efficiency to aggrecan. It was further established that the major site of interaction between COMP and aggrecan lies in the C-terminal domain and this binding was not

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affected by the presence or absence of Ca2+ ions. Additionally, the binding of the C-terminal domain of COMP to aggrecan was higher than thatobserved for full-length COMP, indicating that the major site of interaction between COMP and aggrecan might lie in the C-terminal domain (Chen et al., 2007). By affinity co-electrophoresis, it was found that COMP binds to GAGs – heparin, chondroitin 4 sulfate, chondroitin 6 sulfate – and that these bindings are Ca2+ dependent. These interactions inhibited the binding of COMP to aggrecan by 25%, indicating that the COMP–aggrecan binding might be mediated via the GAG chondroitin sulfate side chains (Chen et al., 2007). In contrast, no binding was observed between COMP and keratan sulfate or heparan sulfate. Interestingly, serum concentrations of COMP as well as GAGs like keratan sulfate and chondroitin sulfate were increased in patients suffering from knee trauma received within 2 months, as compared to older knee traumas (Wakitani et al., 2007). In summary, the binding of COMP to the proteoglycan aggrecan is mediated by GAG — i.e heparin, chondroitin 4 sulfate and chondroitin 6 sulfateside chains, in a dose dependent manner in the presence of Ca2+. While overall COMP–aggrecan binding depends on the presence of Ca2+, the Cterminal domain of COMP binds to aggrecan independent of Ca2+. 3.3. Interaction with fibronectin Fibronectin is an ECM component and is known to be responsible for the adhesion of cells to the surface of the ECM. It has a modular structure and has several functional domains, which can be easily separated by proteolysis. These domains retain their functionality even after separation, and are also capable of influencing the other domains to modulate cell activities (Johnson et al., 2004). Fibronectin binds to COMP in a dose dependent manner and reaches saturation at 1 nM of COMP (Di Cesare et al., 2002). This binding improves significantly in the presence of a specific divalent cation like Mn2 +. Binding studies revealed that COMP shows higher binding to the 70 kDa fragment of fibronectin than full-length fibronectin. Preincubation of COMP with a blocking antibody specific for its globular C-terminal region inhibits binding to fibronectin almost completely, suggesting that binding occurs through the C-terminal domain. Immunohistochemistry revealed that COMP and fibronectin are co-localized in specific punctate areas on the cell surface of chondrocytes that also corresponds to the location of integrins (Di Cesare et al., 2002). Therefore, fibronectin binds to COMP mainly in the C-terminal domain, and specific affinity exists for certain domains of fibronectin (70 kDa) compared to the full length molecule. The effects of this binding interaction on matrix integrity and cellular activity remain largely unknown. 3.4. Interaction with matrilin Matrilins are ECM proteins characterized by having subunits containing von Willebrand factor A (vWFA)-like domains that are connected by EGF-like repeats and a series of heptad repeats at the Cterminal end (Piecha et al., 2002). It was observed that among most ECM components, matrilins-1, -3, and -4 showed the highest binding affinity to COMP. Gel filtration studies showed that these proteins are of similar molecular size and conformation and co-immunoprecipitate, in the presence or absence of Ca2+. Experiments involving truncations of matrilins binding to full length COMP showed that it was mainly the full-length matrilin-4 that bound to COMP, both when it was added in the fluid or in the soluble phase. When either full-length pentameric or monomeric COMP was immobilized on the ELISA plates, only full-length pentameric COMP bound matrilin-4. However, when coated matrilin-4 was able to bind pentameric and monomeric soluble COMP, though the latter was with much decreased affinity. This preference of the oligomeric forms for binding indicates a multivalent binding (Mann et al., 2004). As mutated COMP with deletion D469 was able to bind to matrilin-4 with equal efficiency, it was evident that matrilin-4 binding to COMP

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plays no role in the manifestation of PSACH phenotype associated with the deletion (Mann et al., 2004). Immunohistochemically, using chondrocytes in 3D culture systems in vitro, it was shown that in chondrocytes associated with all the deletions in the COMP molecule associated with PSACH syndrome, namely G427E, D469del, and D511Y-, COMP distribution in the matrix was diffuse and almost absent at 2 weeks and started appearing faintly at 4 weeks (Hecht et al., 2005). In contrast, the control chondrocytes showed heavy staining of the lacunae of the nodules from the beginning and strong COMP distribution in the matrix at 4 weeks. Such a feature was repeated while staining with matrilin-3 and type IX collagen antibodies, where matrilin-3 and type IX collagen expression in the mutations were decreased markedly as compared to control (Hecht et al., 2005). Additionally, immunocolocalization studies revealed that matrilin-3 colocalizes with COMP (Hecht et al., 2005). Although type II collagen expression in these mutations associated with PSACH is unaltered, organized collagen fibrils do not form and the ECM is disorganized. These studies indicated that the combined absence of COMP, matrilin-3 and type IX collagen causes dramatic changes in the matrix and therefore, these proteins play a very significant role in the ECM assembly (Hecht et al., 2005). Transgenic and knockout mouse models for type IX collagen, COMP, and matrilin3, and combinations of these, exhibit cartilage abnormalities and pathologies of varying severities (Zaucke and Grässel, 2009). Matrilins are therefore very important proteins, which along with COMP and type IX collagen play a significant role in cartilage ECM assembly. Certain mutations in COMP molecules are accompanied with impaired secretion of matrilin-3, causing disorganization of the matrix and manifestations of PSACH condition (Hecht et al., 2005; Schmitz et al., 2006; Zaucke and Grässel, 2009). In summary, the binding of COMP to extracellular matrix proteins is essential for the maintenance of the integrity of cartilage and perhaps other extracellular matrices. Mutations in COMP impair the secretion of these ECM proteins and the integrity of the resulting extracellular matrix, in many cases leading to physical abnormalities like PSACH and MED of differing severities. 4. Interaction of COMP with cell surface proteins In addition to binding to components of the extracellular matrix, COMP also binds to cell surface receptors that influence various cellular activities. Cell surface receptors are proteins located on the surface of the cells and are involved in essential functions like cell adhesion, migration, and intracellular communication. The discovery of TSP1 binding to specific cell surface molecules in a saturable manner provided the first evidence that TSPs might regulate cell behavior through direct interactions with cell surface receptors to activate intracellular signaling pathways (Asch et al., 1987; Murphy-Ullrich and Mosher, 1987; Murphy-Ullrich and Iozzo, 2012). Integrins are an important family of cell surface receptors involved in the binding with TSP-1 and 2 due to the presence of RGD- or integrin recognition sequences in their calcium binding domains (Kvansakul et al., 2004). It has been observed that deficiency in the α1 integrin subunit in chondrocytes in mice is associated with the disruption of cartilage homeostasis and an age-dependent development of OA (Zemmyo et al., 2003). COMP possesses an RGD motif within the type 3 calcium binding repeats. Though largely unknown, it was believed that this motif could play a role in chondrocyte attachment. It was shown by DiCesare et al (1994) (DiCesare et al., 1994) that COMP promotes chondrocyte attachment. It was observed that although both the pentameric and reduced forms of COMP promote chondrocyte attachment, the attachment was enhanced when in the reduced state. Also, the pre-incubation of chondrocytes with RGD peptides inhibited this attachment. This indicated that cells interacted with COMP via cell-surface integrin receptors. Among other integrins expressed by chondrocytes, human articular chondrocytes are known to express substantial quantities of α1β1, α5β5 and α5β1 integrins and lesser quantities of α3β1 and α5β3

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heterodimers (Woods et al., 1994). Chondrocyte attachment was inhibited when function-blocking antibodies against β1 sub unit and α5 sub unit were used (α5β1 is expressed substantially by chondrocytes). Antibodies against other integrin subunits did not affect cell attachment indicating that chondrocyte attachment to COMP is mediated by the α5β1 integrin subunit. It was observed that α5β3 integrin (a lesser expressed integrin) subunit played no role in this process. However, on reduction and calcium depletion, the function-blocking antibody against α5β3 integrin subunit was capable of reducing chondrocyte attachment, indicating that COMP utilizes different integrin receptors under different conformations (Chen et al., 2005). All these studies demonstrating the interaction of COMP with different integrin subunits indicate that the RGD motif of COMP is active. Crystal structure of the C-terminal domain of COMP revealed that R367 in the RGD motif is solvent exposed, while G is fully buried and D is responsible for chelating the Ca2+ ion via a bridging water molecule. A G368D mutation (within the RGD motif) impairs the correct folding as well as disrupts the potential interaction with integrins. Additionally, missense mutations among glycine residues in adjoining areas also cause PSACH or MED pathologies. While G366R mutation does not cause structural changes, however, this G is a neighbor of R367GD in the symmetric DGRGD motif. As arginine possesses a bulky, positively charged side chain, this amino acid substitution adjoining the RGD motif might be responsible for disrupting the binding with integrins (Tan et al., 2009). CD47, also known as integrin-associated protein, is a membrane spanning glycoprotein, originally isolated with integrin αvβ3 which functions as an intercellular signaling molecule and as an extracellular ligand for myeloid cells (Brown and Frazier, 2001). It was observed that the α5β3 integrin subunit and CD47 (integrin associated protein) play an important role in ligament cell attachment to COMP. Pre-incubating ligament cells with CD47 antibody at different concentrations demonstrated a dosedependent inhibition of cell attachment. An α5β3 integrin antibody decreased attachment to very modest levels, by about 65%. Immunostaining showed that ligament cells attached to COMP and exhibit fascin localization in microspikes radiating from the tips of cellular protrusions that formed in cells. Addition of PMA (inducer of phosphorylation of fascin and inhibitor the actin-bundling activity) showed that the fascin localization to microspikes was no longer visible and fascin was redistributed to a perinuclear region. Parallel actin staining using phalloidin revealed that a change in fascin distribution was accompanied by decrease in actin microspikes and redistribution of actin into a radial pattern (Rock et al., 2010). These results implicate signal-transduction complexes that include fascin and actin, connected via integrins and CD47 to extracellular COMP. Further evidence of COMP interacting with integrin subunit was observed in vascular smooth muscle cells. It was seen that α7β1 integrin coimmunoprecipitated with COMP in normal rat vascular smooth muscle cells. Neutralizing antibody or siRNA against α7 integrin inhibited vascular smooth muscle cell adhesion to COMP, thereby preventing COMP from its normal function of conserving the contractile phenotype (Wang et al., 2010). Cell surface receptor-COMP binding plays an important role for cellular attachment in primary chondrocytes, immortalized chondrocyte lines, ligament cells and vascular smooth muscle cells (DiCesare et al., 1994; Woods et al., 1994; Brown and Frazier, 2001; Chen et al., 2005; Rock et al., 2010). Different subunits of integrins and CD47 have been demonstrated to take part in this process. The modular pentameric structure of COMP might permit simultaneous binding of both cell-surface proteins and extracellular matrix components. This property may be important for its role in controlling the behavior of target cells, as has recently been described for other multivalent bioconjugates (Conway et al., 2013). 5. Interaction of COMP with extracellular proteases A major phenomenon characterizing arthritis is the breakdown of ECM. Degraded fragments of COMP are found in the cartilage, synovial fluid, and serum of arthritis patients. While the molecular mechanisms

of COMP degradation remain largely unknown, it is plausible that the inhibition of COMP-degrading enzymes can slow down or block the initiation and progression of arthritis. Matrix metalloproteinases (MMPs) are one class of enzymes responsible for degrading the cartilage ECM and releasing fragments of COMP and other ECM components into synovial fluid and serum. A subset of MMPs are stimulated by interleukin-1 (IL-1). Stimulation of nasal cartilage cultures by IL-1 caused proteoglycan release in the first week of culture, COMP release only in the second week, while type II collagen shows almost negligible release till three weeks of culture using nasal cartilage cultures. In articular cartilage, there was a significant release of COMP in the presence or absence of IL-1α stimulation. In the presence of MMP inhibitors, there was only partial inhibition of the COMP release, indicating that other proteases are also involved in the ECM degradation process associated with arthritis (Dickinson et al., 2003). The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) protease family mainly consists of secreted zinc metalloproteinases with a precisely ordered modular organization that includes at least one thrombospondin type I repeat (Apte, 2004; Porter et al., 2005). Member of the ADAMTS family of proteases, including ADAMTS-1, ADAMTS-4, ADAMTS-5, and ADAMTS-8, can degrade cartilage proteoglycans and aggrecans in arthritis. Two closely related members of this family, ADAMTS-7 and ADAMTS-12, were found to bind to COMP. In both cases, the thrombospondin type I repeats at the C-terminal of the protease bind to the EGF-like domain of COMP (Liu et al., 2006a,b). Another member, ADAMTS-4, was also found to degrade COMP to generate a 110 kDa fragment. When purified COMP was incubated at 37 °C with ADAMTS-4, the proportion of the 110 kDa COMP fragment increased in a time-dependent manner. The production of this fragment was completely inhibited when ADAMTS-4 was preincubated with ADAMTS inhibitors (Dickinson et al., 2003) like BB-16 and epigallocatechin gallate (Malfait et al., 2002; Vankemmelbeke et al., 2003). These results demonstrated that COMP can be a substrate for the ADAMTS family of metalloproteinases. When articular cartilage was digested with MMP-3, -12 or -13, and ADAMTS-4 or -5, fragments of COMP were created, while other MMPs like MMP-2, -8, and -9 did not generate any COMP fragments (Zhen et al., 2008). Some earlier reports stated that in a bovine system, MMP-9 was capable of cleaving recombinant as well as purified COMP to generate fragments (Ganu et al., 1998). MMP-19 (found in synovium of normal and rheumatoid arthritis patients) and MMP-20 (found in tooth enamel)

Fig. 1. Extracellular protease MMP-13 degrades COMP. Recombinant COMP (lane a), COMP incubated with recombinant MMP-13 for 2h (lane b), or synovial fluid from osteoarthritic patients undergoing knee replacement surgery (lanes c-i). (A) On probing with anti-MMP-13 antibody, a band corresponding to recombinant MMP-13 (lane b) is also seen in the synovial fluids of all seven OA patients (lanes c-i). (B) On re-probing the same blot with anti-COMP antibody, undigested COMP showed no degradation (lane a), while ~85kDa and ~49kDa fragments were seen in recombinant COMP digested with MMP-13 (lane b). A similar ~85kDa fragment was present in synovial fluid of all 7 OA patients (lanes c-i indicated by arrow).

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Fig. 2. Effect of COMP on human bone marrow mesenchymal stem cells (hBMSCs). (A) Independent culture plates of hBMSCs were transduced with lentiviruses (i) for the overexpression of full length COMP, (ii) carrying the siRNA against full length COMP and (iii) carrying eGFP as transduction control. Media from these cells were collected and subjected to Western Blotting using polyclonal Rabbit anti-COMP antibody. Lane (a) is media from BMSCs overexpressing 6His-tagged COMP, and shows the recombinant COMP migrating slightly slower than the endogenous COMP. Lane (b) is media from cells with siRNA COMP showing reduced COMP expression. Lane (c) is media from untransduced BMSCs and shows endogenous COMP expression. Lane (d) is media from BMSCs overexpressing eGFP and shows similar endogenous COMP expression. (B) Macroscopic observation of micromass pellet cultures after 14 days of incubation in chondrogenic medium. Pellets from cells with elevated COMP, either from lentiviral overexpression or addition of exogenous COMP, were larger than pellets with siRNA–COMP or no exogenous COMP.

were observed to cleave COMP, generating a major 60 kDa fragment, indicating further interaction of COMP with metalloproteases (Stracke et al., 2000). A study in our laboratory was designed to gain insight into the breakdown of COMP by MMP13 in osteoarthritis (Fig. 1). An MMP-13immunoreactive band is present in the synovial fluid of osteoarthritic patients undergoing total knee replacement surgery (Fig. 1a, lanes c–i). We found that recombinant human MMP-13 cleaves recombinant human COMP to generate two major fragments of ~85 kDa and ~49 kDa (Fig. 1B, lane b). On re-probing the same blot with anti-COMP antibody, we found

that the ~85 kDa COMP fragment generated by MMP-13 is also present in synovial fluids from all seven OA patients studied (Fig. 1B (lanes c–i). This elaborates our information about the proteases involved in OA and their correlation with binding to COMP, and indirectly suggests that MMP-13 may in part be responsible for COMP degradation in osteoarthritis. This observation is also supported by an earlier report on cleavage of COMP by MMP-13 (Ganu et al., 1998). In summary, many extracellular proteases of the MMP and ADAMTS families that are involved in the degradation of articular cartilage also generate proteolytic fragments of COMP. These COMP fragments may serve as indicators of OA or the progression of arthritic diseases. 6. Interaction of COMP with hydrophobic compounds

Fig. 3. Distribution of COMP in human cartilage. Cartilage from (A) healthy and (B) OA patient show differences in the quantity and distribution of COMP. On probing, these tissues with anti-COMP antibody, healthy cartilage shows a denser and darker staining, indicating higher COMP deposition. OA cartilage shows lower accumulation of COMP, especially in the deeper zones of the cartilage. Images produced in collaboration with Dr. Cathy S. Carlson.

As stated earlier, the coiled-coil domain of COMP, apart from mediating pentamerization of COMP, allows the binding of different hydrophobic molecules. While some of these molecules might not be of physiological relevance, it is important to study their interaction with COMP to elucidate the structural significance of the coiled-coil domain. Following studies on the crystal structure of the recombinant coiled-coil domain of human COMP (COMPcc) (Efimov et al., 1996; Malashkevich et al., 1996), potential binding molecules in the axial pore of COMPcc were investigated. Among the hydrophobic compounds used in these studies, the most prominent were all-trans retinol, Vitamin D3, benzene, elaidic acid and cyclohexane. Circular dichroism and fluorescence titration experiments confirmed the binding interactions, and suggested that all-trans retinol has a slightly higher binding affinity to COMPcc. X-ray crystallography studies reveal a hydrophobic interaction between COMPcc and all-trans retinol (Guo et al., 1998). Both all-trans retinoic acid and Vitamin D3 signal important events during morphogenesis and repair of bone and cartilage. The ability of COMP to bind these (and other) hydrophobic ligands suggests a storage and delivery function of COMP protein for carrying signaling molecules within a cartilaginous matrix (Özbek et al., 2002), although this requires further investigation.

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Fig. 4. Schematic representation of COMP binding with different proteins. Broken lines represent COMP binding to the proteins, while solid lines represent COMP affecting the proteins within the cell or attached to the membrane receptors.

7. Interaction of COMP with the complement system The complement system is a part of the immune system which aids in the removal of dying cells and immune complexes as well as defends against pathogens (Happonen et al., 2012a). COMP is the first extracellular matrix protein shown to have an active role in inflammation in vivo (Happonen et al., 2010). By binding assays, COMP was seen to activate the alternate pathway by resulting in the deposition of complement C3b and C9. COMP was shown to inhibit the lectin or classical pathways. Experiments showed that the inhibition of the classical pathway occurred at the level of C1q deposition as a result of COMP binding to collagenous stalk of C1q. However, COMP did not bind to the intact C1 complex. COMP also bound to properdin and mannose binding lectin (MBL). Solid phase ELISAs immobilizing different domains of COMP showed that properdin has two binding sites to COMP — one in the TSP-3 like domain and the other in the C-terminal globular domain. MBL and C1q showed binding to all COMP constructs with the C-terminal domain. Binding studies show that the interaction between C3 and COMP is mediated by the C-terminal domain of COMP, which was confirmed by electron microscopy. COMP–C3b complexes were found in the serum of all rheumatoid arthritis (RA) patients, while the level was extremely low for OA patients. However, no difference was found in the levels of COMP–C3b complexes in the synovial fluid in OA and RA patients (Happonen et al., 2010). It was also observed that on treatment with tumor necrosis factor (TNF) α-inhibitors, the level of COMP– C3b in the serum decreased in RA patients, thereby re-confirming that COMP–C3b levels are correlated with inflammatory responses in RA (Happonen et al., 2012b). In a study on arthritis in mice and its immune system activation, it was observed that the titer of anti-COMP antibodies was higher in COMP-deficient mice as compared to COMP-sufficient mice of the

same litter, when immunized with rat COMP. Also, COMP-sufficient mice had lower number of B-cells secreting COMP-reactive antibodies, indicating the development of greater B-cell tolerance to COMP as a result of endogenous COMP, as opposed to lower number of similar B-cells in COMP-deficient mice. These findings, taken together with the fact that COMP-deficient mice did not develop arthritis, point toward the fact that susceptibility to arthritis might be COMP-specific (Geng et al., 2013). Activation of the complement system by COMP is a very interesting recent finding, showing the relevance of released COMP fragments in the serum of RA patients. These studies pave the way for future investigations involving ECM degradation and their inflammatory role in joint diseases. 8. Interaction of COMP with growth factors Growth factors promote cell differentiation, proliferation, and maturation (Patt and Houck, 1983). Growth factors are associated with the initiation and development of long bones of the skeleton, and have important roles in cartilage and other connective tissues (DeLise et al., 2000). Studies with different doses of growth factors like TGF-β2 (Transforming Growth Factor-Beta 2), FGF-2 (Fibroblast Growth Factor-2), and IGF-1 (Insulin-like Growth Factor-1) have shown that while in monolayer condition mesenchymal stem cells show the highest proliferation rates in the presence of 5 ng/ml FGF-2; a remarkable chondrocyte-like differentiation is seen in three-dimensional pellet culture involving 5 ng/ml TGFβ2. The addition of 100 ng/ml of IGF-1 causes a notable increase in the overall proteoglycan synthesis of the pellets. The addition of these growth factors also elicits elevated mRNA and protein levels of COMP (Im et al., 2006). COMP, apart from being one of the important proteins in a cartilaginous ECM, exhibits a

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Fig. 5. Schematic representation of (A) the domains of COMP interacting with different proteins, and their functional roles (adapted from Ullrich-Murphy JE and Iozzo RV, 2012).

wide binding repertoire with other proteins and is involved in the process of chondrogenesis in vitro. In the presence of BMP-2 induction, retroviral over-expression of COMP in murine C3H10T1/2 mesenchymal cells resulted in slightly higher chondrogenesis as compared to uninfected C3H10T1/2 cells. It was also observed that in high density micromass cultures of these mesenchymal cells, the sulfated proteoglycans increased from day 4 to day 10 in COMP expressing BMP-2 induced cultures as compared to untransduced BMP-2 induced cultures. Metabolic sulfate incorporation over a period of 24 h on day 16 of micromass culture revealed a higher 35SO4 content in COMP expressing BMP-2 induced cultures than that seen for untransduced BMP-2 induced cultures or only-COMP expressing cultures (Kipnes et al., 2003). The results from these researchers provided the first evidence indirectly hinting that an interaction may occur between BMPs and COMP. In another report involving vascular smooth muscle cells, it was observed that COMP bound BMP-2 at the C-terminus, inhibited BMP-2 receptor binding and blocked BMP-2 mediated osteogenesis, thereby preventing the calcification of vascular smooth muscle cells (Du et al., 2011). In our laboratory, we studied the role of COMP in chondrogenic pellet culture assay of adult human bone marrow mesenchymal stem cells (hBMSCs). Lentiviral overexpression of COMP in the hBMSCs resulted in larger pellet size compared to the uninfected BMSCs, after two weeks of incubation in chondrogenic media. In contrast, hBMSCs which were infected with siRNA against COMP showed significantly smaller pellet size (Fig. 2). In cartilage tissue isolated from healthy and OA patients, it was observed that five times the normal level of COMP was produced by chondrocytes adjacent to the site of injury or defect as compared to the areas which appeared healthy macroscopically (Koelling et al., 2006). These evidences not only point toward the influence of COMP in chondrogenesis in vitro but also indicate the role played by COMP in the pathogenesis of OA as a factor secreted by chondrocytes to balance against the matrix breakdown (Koelling et al., 2006). Evidence from our laboratory shows that in the cartilage ECM,

the quantity of COMP deposition and its distribution in healthy human (Fig. 3A) are markedly higher and denser, respectively, than that in OA patient (Fig. 3B). This could be associated with the lowering of TGF-β1 signaling with advancing age in OA patients (van der Kraan et al., 2012). These studies paved the way for in-depth research on the interaction(s) of COMP with growth factors which are associated with cartilage/chondrocyte degeneration or maturation (Kawamura et al., 2012; Solorio et al., 2012). COMP–TGF-β binding: Recent studies have shown that COMP directly binds members of the TGF-β superfamily of proteins, namely Bone Morphogenetic Protein (BMP)-2, BMP-4, and BMP-7 as well as TGF-β1 (Haudenschild et al., 2011). COMP–BMP-2 binding promotes osteogenesis in human mesenchymal stem cells by enhancing BMP-2induced intracellular signaling through Smad proteins and increased the levels of BMP receptors. This binding also up-regulated the luciferase activities from a BMP-2-responsive reporter construct, indicating transcriptional activation of osteogenic pathways. In addition to these in vitro effects, COMP–BMP-2 binding also enhanced BMP-2 dependent ectopic bone formation in rat (Ishida et al., 2013). A report elaborating COMP as a primary TGFβ response gene showed that COMP mRNA expression can be detected in high density micromass pellet cultures of human mesenchymal stem cells within a few hours of treatment with TGFβ1. This expression precedes even the expression of type II collagen mRNA, which is a well-known marker for chondrogenesis (Li et al., 2011). These studies suggest that there is COMP–TGFβ1 binding that could influence in vitro chondrogenesis. COMP–TGFβ1 interactions were studied and it has been found that this interaction increases not only the transcriptional activity of mink lung cell line stably transfected with a TGFβ dependent promoter, but also the expression of thrombospondin-1 mRNA in human mesenchymal stem cells (Haudenschild et al., 2011). Ongoing studies in our laboratory will aim at mapping the TSP-like domain of COMP to reveal the influence in binding to TGF-β1, and in chondrogenesis of human

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mesenchymal stem cells (manuscript in preparation). These studies may add insight into the role of COMP in the cellular responses to TGF-β family of ligands, as has been studied for TSP1 (Sweetwyne and Murphy-Ullrich, 2012), as well as the effects of proteolytic degradation of COMP during arthritis. 9. Conclusions COMP binds to a wide variety of ECM proteins, and various other proteins like MMPs are known to regulate the levels of COMP under different conditions. To summarize these interactions, a schematic representation of COMP binding to the different proteins of the cartilage is shown in Fig. 4. The specific domain(s) of COMP that interact(s) with these proteins is shown in Fig. 5, along with their functional role(s). Fig. 5 also shows the exact subunit(s) of the protein which interact with the specific domain(s) of COMP. It is interesting to note that while mutations in the type 3 thrombospondin-like domain of COMP cause severe deformities leading to PSACH and MED syndromes in human, COMP knock-out mice show a normal skeletal phenotype and development (Svensson et al., 2002). Since in vitro chondrogenesis of mesenchymal stem cells is affected by COMP (Haleem-Smith et al., 2012), the compensatory mechanism existing in COMP-null conditions remain to be elaborated. Moreover, while COMP maybe functionally redundant in cartilaginous tissue, pathological conditions like PSACH and MED are not primarily caused by a reduction of COMP in the extracellular matrix, but instead by the effects of a secreted mutated COMP protein (Svensson et al., 2002). The current studies in our laboratory aim at studying the mechanisms by which ECM components like COMP alter cellular responses to growth factors. These investigations may lead to the elucidation of the mechanism of COMP–growth factor interactions in developmental stages in vivo. Acknowledgments We acknowledge Huan Li for the help with the manuscript, and a collaboration with Cathy S. Carlson for the immunohistochemistry shown in Fig. 3. References Adams, J.C., 2001. Thrombospondins: multifunctional regulators of cell interactions. Annu. Rev. Cell Dev. Biol. 17, 25–51. Agah, A., Kyriakides, T.R., Lawler, J., Bornstein, P., 2002. The lack of thrombospondin-1 (TSP1) dictates the course of wound healing in double-TSP1/TSP2-null mice. Am. J. Pathol. 161, 831–839. Agarwal, P., Zwolanek, D., Keene, D.R., Schulz, J.N., Blumbach, K., Heinegard, D., Zaucke, F., Paulsson, M., Krieg, T., Koch, M., Eckes, B., 2012. Collagen XII and XIV, new partners of cartilage oligomeric matrix protein in the skin extracellular matrix suprastructure. J. Biol. Chem. 287, 22549–22559. Apte, S.S., 2004. A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motifs: the ADAMTS family. Int. J. Biochem. Cell Biol. 36, 981–985. Arber, S., Caroni, P., 1995. Thrombospondin-4, an extracellular matrix protein expressed in the developing and adult nervous system promotes neurite outgrowth. J. Cell Biol. 131, 1083–1094. Asch, A.S., Barnwell, J., Silverstein, R.L., Nachman, R.L., 1987. Isolation of the thrombospondin membrane receptor. J. Clin. Invest. 79, 1054–1061. Briggs, M.D., Chapman, K.L., 2002. Pseudoachondroplasia and multiple epiphyseal dysplasia: mutation review, molecular interactions, and genotype to phenotype correlations. Hum. Mutat. 19, 465–478. Briggs, M.D., Hoffman, S.M.G., King, L.M., Olsen, A.S., Mohrenweiser, H., Leroy, J.G., Mortier, G.R., Rimoin, D.L., Lachman, R.S., Gaines, E.S., Cekleniak, J.A., Knowlton, R.G., Cohn, D. H., 1995. Pseudoachondroplasia and multiple epiphyseal dysplasia due to mutations in the cartilage oligomeric matrix protein gene. Nat. Genet. 10, 330–336. Brown, E.J., Frazier, W.A., 2001. Integrin-associated protein (CD47) and its ligands. Trends Cell Biol. 11, 130–135. Budde, B., Blumbach, K., Ylostalo, J., Zaucke, F., Ehlen, H.W.A., Wagener, R., Ala-Kokko, L., Paulsson, M., Bruckner, P., Grassel, S., 2005. Altered integration of matrilin-3 into cartilage extracellular matrix in the absence of collagen IX. Mol. Cell. Biol. 25, 10465–10478. Carlsen, S., Nandakumar, K.S., Backlund, J., Holmberg, J., Hultqvist, M., Vestberg, M., Holmdahl, R., 2008. Cartilage oligomeric matrix protein induction of chronic arthritis in mice. Arthritis Rheum. 58, 2000–2011. Carlson, C.B., Bernstein, D.A., Annis, D.S., Misenheimer, T.M., Hannah, B.L., Mosher, D.F., Keck, J.L., 2005. Structure of the calcium-rich signature domain of human thrombospondin-2. Nat. Struct. Mol. Biol. 12, 910–914.

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