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MATBIO-01067; No of Pages 7 Matrix Biology xxx (2014) xxx–xxx

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

Review

Targeting the extracellular matrix: Matricellular proteins regulate cell–extracellular matrix communication within distinct niches of the intervertebral disc Jake Bedore a, Andrew Leask a,b, Cheryle A. Séguin a,⁎ a b

Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada Department of Dentistry, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada

a r t i c l e Available online xxxx Keywords: Matricellular proteins Intervertebral disc Disc degeneration Extracellular matrix Microenvironment

i n f o

a b s t r a c t The so-called “matricellular” proteins have recently emerged as important regulators of cell–extracellular matrix (ECM) interactions. These proteins modulate a variety of cell functions through a range of interactions with cell-surface receptors, hormones, proteases and structural components of the ECM. As such, matricellular proteins are crucial regulators of cell phenotype, and consequently tissue function. The distinct cell types and microenvironments that together form the IVD provide an excellent paradigm to study how matricellular proteins mediate communication within and between adjacent tissue types. In recent years, the role of several matricellular proteins in the intervertebral disc has been explored in vivo using mutant mouse models in which the expression of target matricellular proteins was deleted from either one or all compartments of the intervertebral disc. The current review outlines what is presently known about the roles of the matricellular proteins belonging to the CCN family, SPARC (Secreted Protein, Acidic, and Rich in Cysteine), and thrombospondin (TSP) 2 in regulating intervertebral disc cell–ECM interactions, ECM synthesis and disc tissue homeostasis using genetically modified mouse models. Furthermore, we provide a brief overview of recent preliminary studies of other matricellular proteins including, periostin (POSTN) and tenascin (TN). Each specific tissue type of the IVD contains a different matricellular protein signature, which varies based on the specific stage of development, maturity or disease. A growing body of direct genetic evidence links IVD development, maintenance and repair to the coordinate interaction of matricellular proteins within their respective niches and suggests that several of these signaling modulators hold promise in the development of diagnostics and/or therapeutics targeting intervertebral disc aging and/or degeneration. © 2014 Published by 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.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CCN proteins: key mediators of nucleus pulposus ECM composition and tissue homeostasis 2.1. CCN2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. CCN1 & CCN3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. SPARC and TSP2: crucial mediators of collagen organization and annulus fibrosus integrity 3.1. SPARC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. TSP2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. New horizons: other matricellular proteins . . . . . . . . . . . . . . . . . . . . . . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Author contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of funding source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competing interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author. Tel.: +1 519 661 2111x82977; fax: +1 519 661 2459. E-mail address: [email protected] (C.A. Séguin).

http://dx.doi.org/10.1016/j.matbio.2014.05.005 0945-053X/© 2014 Published by 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/).

Please cite this article as: Bedore, J., et al., Targeting the extracellular matrix: Matricellular proteins regulate cell–extracellular matrix communication within distinct niches of the intervertebral disc, Matrix Biol. (2014), http://dx.doi.org/10.1016/j.matbio.2014.05.005

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J. Bedore et al. / Matrix Biology xxx (2014) xxx–xxx

1. Introduction Matricellular proteins were initially classified by Bornstein (1995) to emphasize that, in spite of not being direct structural components of the extracellular matrix (ECM), they demonstrate dynamic roles in mediating communication between cells and the surrounding microenvironment. Importantly, matricellular proteins typically exhibit context-specific effects (Roberts, 2011). Conditions that influence the function of matricellular proteins include local variations in growth factor availability, ECM composition and cell-type specific surface receptor expression. As such, matricellular proteins are crucial regulators of cell phenotype and, consequently, tissue function. Given their key role as essential signaling modifiers, the expression of matricellular proteins is tightly regulated; they are typically induced transiently during development or in response to injury or cellular stress, or expressed in a sustained manner in certain chronic pathologies (Bornstein, 2009; Chen and Lau, 2009). Indeed, it is being increasingly recognized that matricellular proteins might represent intriguing targets to treat chronic connective tissue diseases (Jun and Lau, 2011; Tai and Tang, 2008). One such connective tissue disease is intervertebral disc (IVD) degeneration, a disease in which changes in cellular composition and tissue structure lead to changes in the mechanical properties of the tissue and eventual degeneration of the disc structure that lies between adjoining vertebrae of the spine (Gopal et al., 2012). As the population ages, the prevalence of back pain associated with IVD degeneration is increasing at an alarming rate, with the most recent systematic review reporting a global lifetime prevalence of 39% (Hoy et al., 2012). This results in an extreme socioeconomic burden, as back pain has been identified as the leading specific cause of years lived with disability in non-fatal health outcomes of disease and injury (Lim et al., 2012). Our ability to effectively treat IVD degeneration, one of the major underlying causes of back pain, is hampered by an incomplete understanding of mechanisms that control disc development, tissue homeostasis and degeneration. The distinct tissue types and microenvironmental compartments that collectively comprise the IVD provide an excellent paradigm to study how matricellular proteins mediate communication within and between adjacent tissues. The intervertebral disc (IVD) is a complex structure composed of three different tissues which function synergistically to fulfill the physiological function of conferring flexibility to the spine and absorbing axial mechanical loading (Adams and Roughley, 2006) (Fig. 1). The cell types present in the IVD differ in their developmental origins; whereas the annulus fibrosus (AF) and cartilage end plates (CEPs) are derived from the embryonic mesenchyme, the nucleus pulposus (NP) is derived from the embryonic notochord (Choi et al., 2008; McCann et al., 2012). The CEPs anchor the disc both superiorly and inferiorly to adjacent vertebral bodies and play a central role in facilitating nutrient transport. The AF, a fibrocartilaginous structure composed of concentric lamellae of type I collagen, provides the IVD with the tensile strength necessary to encapsulate the NP (Bogduk, 1997). The NP consists largely of a proteoglycan-rich gel that is supported by an irregular network of type II collagen and elastin fibers. The predominant proteoglycan of the disc is aggrecan, which provides osmotic properties to the NP that enable it to maintain turgor against compressive loads, due to the net negative charge of its glycosaminoglycan side chains (Adams and Roughley, 2006; Watanabe et al., 1998). In contrast to the AF and CEPs, whose cellular compositions remain invariant throughout life, the cellular content of the NP changes during development and aging, as large vacuolated notochordal cells are gradually replaced by smaller cartilage-like cells of notochordal origin (Choi et al., 2008; McCann et al., 2012). As is the case with most tissues, maintenance of IVD health is regulated in part via interactions between IVD cells and their extracellular milieu. In recent years, the development of therapies to treat disc degeneration has focused in large part on targeted delivery of growth factors and/or cytokines via intradiscal injection (An et al., 2005;

Fig. 1. Structure and composition of the intervertebral disc. Representative histological section of an intervertebral disc from a 4 week old mouse stained with safranin-O/fast green demonstrates IVD morphology and regionalization of the nucleus pulposus (NP; outlined in black), the annulus fibrosus which is subdivided into inner annulus fibrosus (IA; outlined in green) or outer annulus fibrosus (OA; outlined in dark blue), and cartilaginous end-plate (CEP; yellow) which anchors the IVD to the adjacent vertebral bones (VB). Proteoglycan content is reflected by red stain. The known expression patterns of matricellular proteins in healthy intervertebral discs (IVDs) are also shown. In healthy mature human and mouse discs, CCN2 is expressed in the NP. In contrast, thrombospondin-2 (TSP-2), periostin (POSTN), tenascin (TN) and SPARC are all strongly expressed in the outer annulus fibrosus, with decreasing expression towards inner annulus fibrosus and NP. Of these, only TSP-2 is not found in the NP. Scale bar represents 50 μM.

Chujo et al., 2006; Li et al., 2004; Miyamoto et al., 2006). In fact, the Federal Drug Administration (FDA) has approved phase I clinical trials for both bone morphogenic protein (BMP)-7 (also known as osteogenic protein-1) and BMP-14 (also known as growth and differentiation factor-5) (Zhang et al., 2011). However, these growth factors are by nature potent and multifunctional. Therefore, one might hypothesize that even if they are delivered to the IVD there might be unintended deleterious consequences not only locally but also on adjacent healthy tissue by altering processes such as inflammation or ossification (Zhang et al., 2011). As matricellular proteins have the ability to alter local endogenous signaling responses to growth factors and cytokines, they may represent exquisitely precise molecular targets for therapeutic intervention. Recently the functions of several matricellular proteins within specific tissues of the intervertebral disc have been explored in vivo (Table 1). The current review outlines what is presently known about the role of the matricellular proteins including the CCN family, SPARC (Secreted Protein, Acidic, and Rich in Cysteine), and thrombospondin (TSP) 2 in regulating intervertebral disc cell behavior and disc tissue homeostasis. We specifically focus on findings from genetically modified mouse models in which the expression of candidate matricellular proteins was deleted from one or all compartments of the intervertebral disc. Furthermore, we provide a brief overview of recent preliminary studies of periostin (POSTN) and tenascin (TN). The literature published to date suggests that each of these signaling modulators holds promise in the development of diagnostics and/or therapeutics targeting the diverse etiologies of intervertebral disc aging and/or degeneration. 2. CCN proteins: key mediators of nucleus pulposus ECM composition and tissue homeostasis The CCN (Cyr61, Ctgf, Nov) family of six secreted matricellular proteins (CCN1–6) influences cellular processes such as adhesion, proliferation, migration and differentiation in a cell type-specific manner (Brigstock, 2003; Perbal, 2004). Accordingly, CCN proteins can modulate the signaling potential of a wide range of extracellular ligands

Please cite this article as: Bedore, J., et al., Targeting the extracellular matrix: Matricellular proteins regulate cell–extracellular matrix communication within distinct niches of the intervertebral disc, Matrix Biol. (2014), http://dx.doi.org/10.1016/j.matbio.2014.05.005

J. Bedore et al. / Matrix Biology xxx (2014) xxx–xxx

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Table 1 Summary of matricellular protein localization and phenotype of the intervertebral disc in knockout mouse models. Matricellular protein

Localization in healthy IVD

Expression changes in diseased states

IVD phenotype of knockout mouse model

References

CCN2

Enriched in the NP, expressed in the AF (mouse)1

Increase in NP with disc degeneration grade (human)2

1

Enriched in the outer AF, gradient of decreasing expression towards the NP (human)4 Enriched in the outer AF, weak expression in the inner AF (human)6 Enriched in the outer AF, gradient towards decreasing expression in inner AF and NP (human)7 Enriched in the outer AF, gradient towards decreasing expression in inner AF and NP (human)9 Enriched in AF, weakly expressed in NP (mouse)10

Decrease with age and degeneration grade in AF (human)4

Reduced aggrecan1,3 and type II collagen expression Accelerated age-related degeneration1 Disruption of proper laminar organization and decreased type I collagen expression in AF5 Disruption of proper laminar organization6

SPARC

TSP-2 PSTN

TN

CCN3

Unknown

Bedore et al. (2013) Tran et al. (2010) 3 Tran et al. (2013a) 4 Gruber et al. (2004) 5 Gruber et al. (2005) 2

6

Gruber et al. (2008)

7

Increase in NP and AF with degeneration grade (human) or needle-puncture injury (rat)8 No change in expression (human)9

Unknown

Unknown

9

Unknown

Unknown

10

including growth factors and ECM components (Brigstock, 2003; Leask and Abraham, 2006). Of the CCN family, CCN1 (Cyr61) and CCN2 (CTGF) appear to possess a similar expression patterns and activities (Moritani et al., 2005). Conversely, CCN3 appears to be expressed reciprocally and to act antagonistically to both CCN1 and CCN2 (Riser et al., 2009, 2010; Tran et al., 2011). Indeed, CCN3 frequently shows opposing expression patterns to those of CCN1 and CCN2 during development (Perbal, 2001) and in vitro (Perbal, 2006). Structural differences and similarities between CCN family members may underlie their respective biological functions (Holbourn et al., 2008). Regulation of the diverse cellular responses mediated by the CCN proteins likely involves a balance between opposing signals induced by different members of the CCN family, which are often co-expressed in the tissue microenvironment.

2.1. CCN2 There is a growing body of evidence that supports the notion that CCN2 plays an important role in regulating cell activity, ECM composition and tissue homeostasis of the IVD, most notably in the nucleus pulposus. During murine embryonic development, expression of Ccn2 has been localized to early node and notochord tissues that will give rise to the nucleus pulposus (Tamplin et al., 2011). In zebrafish, proximal Ccn2 promoter activity was observed in the notochord during embryonic development, and knockdown of Ccn2 expression led to early embryonic lethality (Chiou et al., 2006). Within the IVD, CCN2 was first identified as an anabolic factor secreted by notochordal cells, capable of inducing NP cell proliferation and aggrecan production in vitro (Aguiar et al., 1999; Erwin et al., 2006; Erwin and Inman, 2006). Studies in cultured nucleus pulposus cells demonstrated that CCN2 expression is induced by TGF-β signaling (via Smad3 and AP-1) (Tran et al., 2010) and that shRNA-mediated Ccn2 suppression decreased type II collagen and aggrecan expression (Tran et al., 2013a) (the molecular regulation of CCN2 expression recently reviewed in (Tran et al., 2013b)). Consistent with in vitro observations, the loss of CCN2 expression by nucleus pulposus cells in vivo is associated with decreased type II collagen content within the IVD (Bedore et al., 2013), as well as a decrease in aggrecan content of the NP in embryonic and newborn mice (Bedore et al., 2013; Tran et al., 2013a). Taken together, these results demonstrate that secretion of CCN2 into the nucleus pulposus tissue microenvironment positively regulates structural components of the ECM that are crucial to the function of healthy nucleus pulposus tissue (Adams and Roughley, 2006; Watanabe et al., 1998). These findings are consistent with in vitro data suggesting that CCN2 can directly induce aggrecan and type II collagen in growth plate and articular chondrocytes (Takigawa, 2013; Takigawa et al., 2003) as well as in vivo data showing increased aggrecan and type II collagen expression

Gruber et al. (2011) Tsai et al. (2013)

8

Gruber et al. (2002)

Tran et al. (2011)

from epiphyseal and articular chondrocytes of cartilage-specific CCN2overexpressing mice (Itoh et al., 2013; Tomita et al., 2013). Recent mechanistic studies have demonstrated that in NP cells, CCN2 forms a negative feedback loop with hypoxia-inducible factor 1 (HIF-1α) (Tran et al., 2013a), which is a known regulator of cell survival, metabolism and ECM synthesis in NP cells (Agrawal et al., 2007; Gogate et al., 2011; Risbud et al., 2006; Zeng et al., 2007). Furthermore, CCN2 also moderates the catabolic effects of IL-β in NP cells through binding of CCN2 to integrins ανβ3 and α5β1 (Tran et al., 2014). Interestingly, a recent study demonstrated differential responses in terms of cell proliferation, proteoglycan secretion and gene expression profiles of human NP cells isolated from either moderately or severely degenerated IVDs to treatment with exogenous anabolic factors, including CCN2 (Abbott et al., 2013). Their findings demonstrate that the grade of tissue degeneration alters the cellular response to CCN2, which induced a catabolic response in cells isolated from discs with advanced degeneration. It is interesting to note that differential expression profiles and altered function of integrins have been reported between non-degenerated and degenerated or herniated discs (Le Maitre et al., 2009; Xia and Zhu, 2008, 2011), which may modulate the effects of CCN2. Importantly, the changes in integrin expression profiles in NP cells may be an example of the change in NP cell composition and/or phenotype that has been postulated to occur throughout development, aging and disease (Pattappa et al., 2012) resulting in changes in extracellular matrix composition and tissue microenvironment. This changing landscape in the IVD makes it difficult to pinpoint the precise factors altering the cellular response to CCN2 and points to the importance of assessing its effects in vivo. These findings provide yet another example of how the function of matricellular proteins within specific tissue niches can be highly variable. Previous studies localized CCN2 expression to degenerated human IVDs and suggested that this increased expression contributed to accelerated disc fibrosis and enhanced disc vascularization (Ali et al., 2008; Peng et al., 2009). In contrast, the association between CCN2 expression and IVD degeneration led others to speculate that CCN2 expression reflects the initiation of a reparative response, triggering ECM remodeling and promoting cellular survival (Croci et al., 2004; Tran et al., 2010). Our recent studies employed a transgenic mouse model with specific deletion of CCN2 in notochord-derived NP cells to address this question. These studies demonstrate that the loss of CCN2 from nucleus pulposus cells accelerates disc degeneration, consistent with the notion that CCN2 expression in the nucleus pulposus promotes the maintenance and repair of the IVD (Bedore et al., 2013). It is tempting to speculate that in the absence of NP-derived CCN2, an increased collagen-to-PG ratio may contribute to altered load transmission during compressive loading of the NP (Hsieh and Twomey, 2010), ultimately leading to a vicious cycle of increased fibrous matrix production and the inability of

Please cite this article as: Bedore, J., et al., Targeting the extracellular matrix: Matricellular proteins regulate cell–extracellular matrix communication within distinct niches of the intervertebral disc, Matrix Biol. (2014), http://dx.doi.org/10.1016/j.matbio.2014.05.005

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J. Bedore et al. / Matrix Biology xxx (2014) xxx–xxx

the tissue to withstand compressive loading. The decreased NP hydration reported in the absence of CCN2 would lead to concomitant NP depressurization and an altered loading profile resulting in AF overloading (increased stress peaks) (Adams et al., 1993), increasing susceptibility of the disc to failure upon future loading (McNally et al., 1993). The increase in disc herniation reported in the absence of NP-derived CCN2 (Bedore et al., 2013) may be a consequence of the altered mechanical environment of the IVD. The anabolic effects of CCN2 in the IVD are consistent with previous observations showing that CCN2 expression is upregulated in osteoarthritic cartilage (Omoto et al., 2004) and the ability of rCCN2 peptide to promote cartilage repair in both the MIA-induced model of osteoarthritis and artificially induced articular cartilage defects (Nishida et al., 2004). Overall, evidence obtained from in vivo animal models supports the parallel protective roles of CCN2 expression in the nucleus pulposus and articular cartilage. Why might we see similar anabolic effects in these tissues? Although the phenotype of NP cells in regard to metabolic activity and gene expression has been described as quite different from that of articular cartilage in studies of young rabbit and sheep (Melrose et al., 2003; Poiraudeau et al., 1999; Steck et al., 2005), human NP cells have been shown to express specific receptors and ECM components with which CCN2 is known to interact. For example, both NP cells and articular chondrocytes produce an aggrecan-rich matrix, and express integrin subtypes through which CCN2 mediates anti-catabolic effects (Tran et al., 2014) such as the classical fibronectin receptor α5β1 (Salter et al., 1992) and αν integrin (Ostergaard et al., 1998). The data collectively suggest that CCN2 is an important regulator of nucleus pulposus ECM composition and health that may represent an intriguing clinical target for delaying the onset of age-associated disc degeneration or promoting the repair of degenerative discs at early stages of disease progression. 2.2. CCN1 & CCN3 Although CCN2 is the most studied member of the CCN family in the IVD, the roles of other CCN proteins in the disc are beginning to be explored. CCN1 shows significant functional similarities to CCN2 (Brigstock, 1999; Brunner et al., 1991; Kubota et al., 2001; Lau and Lam, 1999). The diverse cellular responses mediated by CCN1 can be ascribed, at least in part, to its role as an inducer of both adhesive and proliferative signaling cascades (Lau, 2011). Similar to CCN2, CCN1 expression is enriched in both the embryonic notochord and notochord-derived NP of the IVD (Bedore et al., 2013). In vitro, CCN1 appears to have similar effects as CCN2 on cultured chondrocytes and osteoblasts (Kawaki et al., 2011; Moritani et al., 2005) and, like CCN2, promotes chondrogenesis in mouse limb bud-derived mesenchymal cells (Wong et al., 1997). These results suggest that CCN1 might act synergistically with CCN2 to contribute to IVD function. However, this hypothesis has yet to be evaluated. Similar to CCN2, the expression and localization of CCN3 within the IVD are age-dependent. Although slightly more restricted, Ccn3 mRNA shows a similar gene expression pattern to Sonic Hedgehog (Shh) during embryonic development, detected along the floor plate and notochord (with a similar left/right asymmetry in the node) (Katsube et al., 2009). In the rat, CCN3 was shown to be robustly expressed in both nucleus pulposus and annulus fibrosus cells in the putative disc during embryonic development, with reduced expression in the adult disc (Tran et al., 2011). In contrast to CCN2, CCN3 appears to be a negative regulator of IVD growth, as treatment of nucleus pulposus cells in vitro with exogenous CCN3 reduced cell proliferation and decreased the expression of ECM components including aggrecan, versican and type I collagen (Tran et al., 2011). Interestingly, our lab has recently reported an expression pattern of CCN3 in the mouse IVD that differs from that previously reported in the rat, but is in keeping with the balanced and reciprocal regulation of CCN2/CCN3 expression and activity seen in cartilage (Kawaki et al., 2008). We demonstrated that in contrast to

the expression of CCN1 and CCN2, CCN3 was not enriched in the developing notochord relative to the adjacent mesenchymal tissue, nor was robust expression detected in mature IVDs (Bedore et al., 2013). 3. SPARC and TSP2: crucial mediators of collagen organization and annulus fibrosus integrity 3.1. SPARC Secreted Protein, Acidic, and Rich in Cysteine, also known as osteonectin, is a calcium-binding protein that is known to modulate cell–ECM interactions through direct binding of ECM components during tissue remodeling. SPARC expression is largely restricted to tissues that undergo consistent turnover such as bone, gut mucosa, healing wounds or tumors (Bradshaw and Sage, 2001; Brekken and Sage, 2000). Consistent with these observations, SPARC plays an important role in modulating collagen fibrillogenesis, deposition and remodeling (Bradshaw and Sage, 2001). Moreover, SPARC modulates growth factor efficacy, interacts with several resident matrix proteins, acts as an antiadhesive factor and modulates the expression of matrix metalloproteinases (MMPs) (Bradshaw and Sage, 2001). Collectively, these findings point to an important role for SPARC in regulating cell–ECM interactions during development and injury response. Interestingly, within the IVD the spatial and temporal patterns of SPARC expression contrast those of CCN2. The expression of SPARC is enriched in the outer annulus fibrosus, with lower expression detected in cells of the inner annulus and nucleus pulposus (Gruber et al., 2004). In contrast to CCN2, whose expression in humans increases with age and severity of degeneration, the expression of SPARC is reported to decrease with age and severity of degeneration (Gruber et al., 2004). Genetically modified mice with whole-body deletion of the SPARC gene demonstrate severe IVD herniations at 14, 19 and 20 months of age that do not occur in wild-type controls (Gruber et al., 2005). In contrast to wild-type mice in which the AF is composed of collagen fibrils of uniform diameter with a regular fibril margin, AF tissues of SPARC-null mice show collagen fibrils that range widely in size and diameter, and possess irregular margins indicative of improper collagen packaging and assembly. Although no overall differences in average fibril size were observed, a marked difference in the pattern of fibril size distribution was reported (Gruber et al., 2005). The precise mechanism underlying the disc failure observed in SPARC-null mice is unclear; however, the down-regulation of type I collagen expression (Gruber et al., 2005) incomplete pericellular pro-collagen processing, and differences in growth factor activity and/or ECM configuration in SPARC-null mice are likely major contributing factors (Bradshaw and Sage, 2001; Juneja and Veillette, 2013). Studies aimed at characterizing and quantifying discogenic pain associated with disc degeneration have also been conducted using the SPARC-null mouse model. SPARC-null mice exhibit age-dependent, region-specific hypersensitivity, stretch-induced axial discomfort, motor impairment and reduced physical function (Millecamps et al., 2012) mirroring symptoms of discogenic pain in humans. Given the similarities between the histological appearance of disc degeneration in SPARC-null mice and human tissues, the SPARC-null mouse provides an interesting model to study the association between changes in IVD tissue composition and histological hallmarks of disc degeneration and chronic back pain. These studies provide yet another example of how matricellular proteins are crucial regulators in IVD health. Further evidence supporting the protective role of SPARC in fibrocartilaginous tissues has been reported in the periodontal ligament, a structurally and functionally similar tissue to the AF in which SPARC plays a major role in tissue health. SPARC-null mice show decreased collagen content in the periodontal ligament (Trombetta and Bradshaw, 2010). In a lipopolysaccharide-induced model of inflammatory periodontal disease, SPARC-null mice also demonstrate a greater loss

Please cite this article as: Bedore, J., et al., Targeting the extracellular matrix: Matricellular proteins regulate cell–extracellular matrix communication within distinct niches of the intervertebral disc, Matrix Biol. (2014), http://dx.doi.org/10.1016/j.matbio.2014.05.005

J. Bedore et al. / Matrix Biology xxx (2014) xxx–xxx

in periodontal ligament collagen content, maxillary bone, and gingival connective tissue compared to wild-type mice (Trombetta-Esilva et al., 2011), suggesting a protective role for SPARC in this connective tissue disease.

3.2. TSP2 The anti-angiogenic matricellular protein thrombospondin (TSP) 2 (Kyriakides et al., 1998; Lawler and Lawler, 2012) plays an important role in the organization of collagen fibrillogenesis in skin and tendon, as demonstrated by studies using TSP2-null mice (Kyriakides et al., 1998). In addition to demonstrating fragile skin with reduced tensile strength and increased blood vessel formation, TSP2-null mice possess an abnormally flexible tail (Kyriakides et al., 1998). Interestingly, increased levels of TSP2 transcript are detected in tendon tissues during embryonic development, which are more cellular and grow rapidly compared to adult tissues (Juneja and Veillette, 2013). Thrombospondin expression in the human IVD is localized to cells of both the inner and outer annulus fibrosus (Gruber et al., 2008). In both human and rat intervertebral discs, TSP2 is strongly expressed in the outer annulus, expressed weakly in the inner annulus, and not expressed in the nucleus pulposus (Gruber et al., 2008, 2006). TSP2 expression in the outer annulus is thought to contribute to the avascular nature of the disc. However, although there was an increase in vascularity at the periphery of the disc in TSP2-null mice, no vascular ingression beyond the outer AF was observed (Gruber et al., 2008). Similar to the SPARC-null mice, disruption of the TSP2 gene results in disruption of annular lamellar organization (Gruber et al., 2008). Variation in collagen fibril size and shape seen in TSP2-null mice has been attributed to elevated levels of MMP2 in the pericellular space, associated with a reduction in transglutaminase II-induced collagen cross-linking (Agah et al., 2005). Although no mention of IVD herniation was made in the study of TSP2-null mice at five months of age, it is tempting to speculate that the alterations observed in lamellar organization would decrease the overall mechanical integrity of the AF and increase the propensity for disc rupture associated with age-related degeneration.

4. New horizons: other matricellular proteins In addition to those described above, several other matricellular proteins demonstrate expression patterns and/or functions that suggest they may also contribute to IVD function. For example, similar to SPARC and TSP2, strong expression of periostin (POSTN) and tenascin (TN) is detected in the outer AF; both show a gradient of decreasing expression towards the inner AF and NP (Gruber et al., 2002, 2011). POSTN gene expression is upregulated in response to increased mechanical loading, and is essential to the integrity and function of the periodontal ligament following tooth eruption during occlusal loading (Rios et al., 2008). When compared to healthy controls, degenerated human discs and rat discs subjected to the needle-puncture model of disc injury show elevated POSTN gene and protein expression in both the NP and AF (Tsai et al., 2013). TN is constitutively expressed in tissues subjected to high levels of mechanical stress such as tendons, ligaments and arterial walls, and is upregulated in a dose-dependent manner in response to increased loading (Chiquet-Ehrismann and Chiquet, 2003). However, in contrast to POSTN, TN expression shows no changes in expression level or localization between control and degenerated human discs (Gruber et al., 2002). Taken together, these data demonstrate the central role played by matricellular proteins in the adaptive response of connective tissues to mechanical loading, making it tempting to speculate that their expression may also contribute to the response of IVD tissues to physiological or pathological loading. The precise roles of POSTN and TN in IVD development, homeostasis, repair and/or degeneration however remain to be elucidated.

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5. Conclusion IVD tissues contain distinct cell types and extracellular matrix compositions that reflect their distinct physiological functions. Moreover, each specific tissue type contains a different matricellular protein composition, which varies based on the specific stage of development, maturity or disease. A growing body of direct genetic evidence links IVD development, maintenance and repair to the coordinate interaction of matricellular proteins within their respective niches in the IVD. Thus matricellular proteins may be prime candidates to target as novel biomarkers or therapeutic targets for the treatment of disc degeneration. However, in humans and in animal models alike, there are numerous factors influencing the function of matricellular proteins in tissue homeostasis that must be considered. First, the ECM composition and mechanobiology of the IVD change progressively through aging and disease. Since context is a crucial determinant of the function of matricellular proteins, it is likely that their physiological and pathophysiological roles in the IVD differ based on the distinct microenvironment of the disc associated with particular phases of development, maturity and disease. Second, the relationship between the tissue composition of the disc and the clinical presentation of symptoms associated with degenerative disc disease is not well understood. It is clear that certain matricellular proteins govern ECM composition and consequently tissue morphology within the intervertebral disc. However, for several of these matricellular proteins, the precise mechanisms responsible for their effects in the disc have yet to be determined. Despite the recent advances that have established a central role for matricellular proteins in governing disc cell biology, the relationship between ECM alterations and the onset of symptomatic disc-related pain remains unknown and warrants further investigation. Author contributions All authors were involved in drafting the article or revising it critically for content, and all authors approved the final version to be published. Role of funding source The Canadian Institutes of Health Research (CIHR) supports both C.A.S. [MOP-115718] and A.L. [MOP-77603]. J.B. is supported by the Joint Motion Program — A CIHR Training Program in Musculoskeletal Health Research and Leadership as well as The Arthritis Society (TAS). C.A.S. is the recipient of a Scholar Award from TAS. Competing interest statement The authors declare that they have no competing interests. References Abbott, R.D., Purmessur, D., Monsey, R.D., Brigstock, D.R., Laudier, D.M., Iatridis, J.C., 2013. Degenerative grade affects the responses of human nucleus pulposus cells to link-N, CTGF, and TGFbeta3. J. Spinal Disord. Tech. 26, E86–E94. Adams, M.A., Roughley, P.J., 2006. What is intervertebral disc degeneration, and what causes it? Spine (Phila Pa 1976) 31, 2151–2161. Adams, M.A., McNally, D.S., Wagstaff, J., Goodship, A.E., 1993. Abnormal stress concentrations in lumbar intervertebral discs following damage to the vertebral bodies: a cause of disc failure? Eur. Spine J. 1, 214–221. Agah, A., Kyriakides, T.R., Bornstein, P., 2005. Proteolysis of cell-surface tissue transglutaminase by matrix metalloproteinase-2 contributes to the adhesive defect and matrix abnormalities in thrombospondin-2-null fibroblasts and mice. Am. J. Pathol. 167, 81–88. Agrawal, A., Guttapalli, A., Narayan, S., Albert, T.J., Shapiro, I.M., Risbud, M.V., 2007. Normoxic stabilization of HIF-1alpha drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat intervertebral disk. Am. J. Physiol. Cell Physiol. 293, C621–C631. Aguiar, D.J., Johnson, S.L., Oegema, T.R., 1999. Notochordal cells interact with nucleus pulposus cells: regulation of proteoglycan synthesis. Exp. Cell Res. 246, 129–137. Ali, R., Le Maitre, C.L., Richardson, S.M., Hoyland, J.A., Freemont, A.J., 2008. Connective tissue growth factor expression in human intervertebral disc: implications for angiogenesis in intervertebral disc degeneration. Biotech. Histochem. 83, 239–245.

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Please cite this article as: Bedore, J., et al., Targeting the extracellular matrix: Matricellular proteins regulate cell–extracellular matrix communication within distinct niches of the intervertebral disc, Matrix Biol. (2014), http://dx.doi.org/10.1016/j.matbio.2014.05.005