The Biology of Small Leucine-rich Proteoglycans in Bone Pathophysiology

Abstract

The class of small leucine-rich proteoglycans (SLRPs) is a family of homologous proteoglycans harboring relatively small (36–42 kDa) protein cores compared with the larger cartilage and mesenchymal proteoglycans. SLRPs have been localized to most skeletal regions, with specific roles designated during all phases of bone formation, including periods relating to cell proliferation, organic matrix deposition, remodeling, and mineral deposition. This is mediated by key signaling pathways regulating the osteogenic program, including the activities of TGF-β, bone morphogenetic protein, Wnt, and NF-κB, which influence both the number of available osteogenic precursors and their subsequent development, differentiation, and function. On the other hand, SLRP depletion is correlated with degenerative diseases such as osteoporosis and ectopic bone formation. This minireview will focus on the SLRP roles in bone physiology and pathology.

SLRP Structure and Classification

The small leucine-rich proteoglycans (SLRPs)² were originally defined as proteoglycans with a relatively small protein core (36–42 kDa) harboring tandem leucine-rich repeats and undergoing post-translational modifications, including substitution with glycosaminoglycan side chains of various types (1, 2). Their ubiquitous tissue distribution and expression at strategic sites in embryogenesis and tissue repair, coupled with their protein conservation, suggests that SLRP functions are of no small consequence. Originally, the SLRPs were grouped into three distinct classes based on nucleotide and protein sequence conservation, the organization of disulfide bonds at their N and C termini, and their genomic organization (2, 3). More recently, the SLRP gene family has expanded to encompass 18 genes classified into five distinct subfamilies (Table 1) (4, 5), additionally based on N-terminal Cys-rich clusters of the protein core and ear repeats (C-terminal repeats specific to SLRPs) (6), chromosomal organization (4, 5), and, importantly, functional commonality in view of the fact that some SLRPs are not classical proteoglycans (4). Thus, the canonical class I members decorin and biglycan contain chondroitin or dermatan sulfate side chains, whereas the more recently described asporin does not (7, 8). On the other hand, all class II members bear keratan sulfate chains or polylactosamine in their leucine-rich repeats, whereas class III members carry keratan sulfate (osteoglycin), chondroitin/dermatan sulfate (epiphycan), or no glycosaminoglycan (opticin) chains (4, 9). However, most non-canonical class IV and V members (10–13) unexpectedly lack any glycosaminoglycan chain, with the exception of chondroadherin, which is substituted with keratan sulfate (10). Therefore, the unique characteristics of their protein cores and the presence of glycosaminoglycans, together with specific post-translational modifications, notably changes in the degree of glycosaminoglycan epimerization or sulfation, characterize this class of proteoglycans with high structural complexity.

TABLE 1.

SLRP classes: organization and interacting partners

SLRP Functional Network

Following synthesis, most of the SLRPs are secreted into the pericellular matrix, where they either diffuse and bind to components of the extracellular matrix (ECM), e.g. collagens, or remain free. The localization of the SLRPs in the ECM appears to be strictly predetermined, with specific SLRPs being predominantly distributed in the ECM “proper” (i.e. further removed from the cell or inter-territorial), whereas others are localized both to the ECM and pericellular matrix (territorial, immediately surrounding cells) (7, 14, 15). In addition, the localization of SLRPs to specific compartments is dependent on the tissue of origin (16, 17). ECM-proper SLRPs bind to various types of collagens, thereby regulating the kinetics, assembly, and special organization of fibrils in skin, tendons, bone, and cornea (Fig. 1A) (2, 18–20). However, besides being sequestered mainly in the ECM, ECM-proper SLRPs can also exist as soluble molecules, e.g. when released from the ECM by proteolytic digestion of injured tissues. Both bound and soluble SLRPs interact with various growth factors, including TGF-β (Fig. 1B) (21), bone morphogenetic protein BMP4 (22), WISP-1 (Wnt1-inducible secreted protein-1) (23), von Willebrand factor (24), PDGF (25), TNF-α (26), and insulin-like growth factor I (IGF-I) 27). This biological interaction modulates growth factor bioavailability through the formation of specific concentration gradients. On the other hand, SLRP pericellular localization allows these molecules to interact with different ligands and cell-surface receptors, thereby modulating a wide range of cell-matrix interactions (4). Pericellular decorin binds the EGF receptor and ErbB4 in tumor cells, leading to transient activation of the MAPK pathway, mobilization of intracellular Ca²⁺ levels, and induction of the cyclin-dependent kinase inhibitor p21^WAF1 (28) with subsequent down-regulation of the receptor (29–31). Furthermore, decorin binds directly to Met, the receptor for hepatocyte growth factor (32), which is noteworthy, as signaling through Met leads to the phosphorylation of β-catenin, a known effector of the crucial Wnt signaling pathway that up-regulates tumor cell motility, tissue invasion, and metastasis.

FIGURE 1.

Schematic representation of the roles of SLRPs and their specific signaling pathways in osteogenesis and remodeling. A, SLRPs have specific roles during all phases of bone formation, including periods relating to cell proliferation, organic matrix deposition, remodeling, and mineral deposition. B, binding of TGF-β to SLRPs (decorin and lumican) regulates its downstream Smad2/3 signaling by facilitating or inhibiting ligand presentation to respective receptors to control bone formation. C, biglycan stimulates ERK phosphorylation and signal transduction through the transcription factor Runx2 to promote osteoblast differentiation. D, biglycan sustains the binding of BMP2/ALK6 to enhance Smad1/5/8 phosphorylation to facilitate downstream signaling relevant to osteogenesis. E, biglycan promotes Wnt3a binding to the LRP6 coreceptor subunit (which engages the Frizzled receptor for complete Wnt-mediated signaling), thereby activating β-catenin signaling to promote bone growth. TCF, T cell factor.

Under physiological conditions, decorin signaling through the IGF-I receptor exerts anti-apoptotic effects, favoring in this manner normal cell growth. By binding to the IGF-I receptor, decorin triggers phosphorylation and downstream activation of phosphoinositide 3-kinase, Akt/protein kinase B, and p21^WAF1, inducing an anti-apoptotic effect (27). Another class I SLRP, biglycan, acts as a signaling molecule important for the innate immune system. Upon tissue stress or injury, sequestered and immunologically inactive biglycan is released from the ECM through a so far undefined proteolytic mechanism. Soluble biglycan interacts with the innate immunity Toll-like receptors TLR-2 and TLR-4 on macrophages, taking on a role of an endogenous ligand and consequently triggering an inflammatory response (33, 34). This role could also be relevant in tumor biology, as active Toll-like receptor signaling in tumor cells was shown to affect transformed cell functions (35, 36). A recent report (37) has shown that also decorin binds to TLR-2/4, thereby inducing the production of PDCD4 (proinflammatory programmed cell death 4) in macrophages. Moreover, decorin prevents the translation repression of PDCD4 via induction of microRNA-21, a PDCD4 repressor. Thus, decorin can boost proinflammatory activity and concurrently retard tumor growth by induction of PDCD4 and microRNA-21.

Additional signaling pathways have also been identified for the class II SLRP, lumican. Fas-FasL signaling, a key cellular pathway, was found to be disrupted in Lum^−/− stromal corneal keratocytes (38). Consequently, it has been proposed that that lumican could directly bind FasL, thereby facilitating induction of Fas. Poor signaling through Fas-FasL in Lum^−/− mice ultimately leads to attenuated apoptosis of stromal cells, impaired induction of inflammatory cytokines, impaired recruitment of inflammatory cells, and retarded corneal healing. Lumican appears to regulate fibroblast proliferation by modulating specific cell growth mediators. Thus, Lum^−/− fibroblasts have decreased p21^WAF1, a universal inhibitor of cyclin-dependent kinases, and a consequent increase in cyclins A, D₁, and E. Furthermore, the tumor suppressor p53, an upstream regulator of p21^WAF1, is down-regulated in Lum^−/− fibroblasts, suggesting regulation of p21^WAF1 by lumican in a p53-dependent manner (38). Lumican overexpression was found to suppress tumorigenic transformation of rat fibroblasts induced by v-src and v-K-ras (39), suggesting that down-regulation of lumican expression may play a role in development of some human cancers. In a recent study by Nikitovic et al. (40), lumican was found to modulate the crucial Smad signaling pathway in bone tumor cells.

Given that SLRP production, turnover, and ultimate localization are dynamic processes, variable SLRP availability at different compartments would facilitate signaling pathways regulating specific biological and pathological processes (41). However, the exact mechanisms regulating equilibrium between “free” and bound SLRPs clearly need to be further studied. Some important questions that need to be answered would be how changes in the ECM composition shunt more bound SLRPs into the free pool and to what extent their release is dependent on the degradation of the retaining ECM. Furthermore, we should also consider the activities of processed SLRP fragments vis-à-vis the parent proteins, especially relevant under pathological conditions. Indeed, significant proteolysis of SLRP protein cores occurs in both normal and arthritic tissues, with specific catabolites of biglycan and fibromodulin identified only in the cartilage from damaged joints (42).

The collective findings summarized above clearly indicate that numerous pathways can be affected by the SLRPs. Whether the same pathways are operative in bone biology and musculoskeletal tissues is not entirely clear at this time. However, these investigations provide a fundamental platform upon which to build and deepen our understanding of SLRP function in bone (Fig. 1B).

Key Roles of SLRPs in Bone Physiology

It is now firmly established that specific SLRPs are functionally involved in normal bone development and homeostasis. Using a combination of in situ hybridization and immunohistochemistry, class I SLRPs (biglycan and decorin) have been localized to many skeletal regions, including articular and epiphyseal cartilage, vascular canals, subchondral regions, and the periosteum (43). SLRPs have specific roles during all phases of bone formation, including periods relating to cell proliferation, organic matrix deposition, remodeling, and mineral deposition (44, 45). Generation of mutant mice with targeted deletion of Bgn has shown that biglycan-deficient mice are viable and have no profound skeletal patterning abnormalities at birth. However, with age, their long bones show decreased length coupled with decreased mineral density and mass compared with age-matched controls (46). Biglycan deficiency leads to structural abnormalities in collagen fibrils in bone, dermis, and tendon and to a “subclinical” cutaneous phenotype with thinning of the dermis but without overt skin fragility (47). Furthermore, male mice depleted of biglycan (the Bgn gene is located on the X chromosome; thus, male mice do not contain a second allele of Bgn), designated Bgn^−/0,+/0, exhibit reduced bone formation compared with control mice, indicating that biglycan is important for the cells that control bone production (47).

The key role of biglycan in bone development is corroborated by the observation that bone marrow ablation in biglycan-null mice leads to delayed osteogenesis (48). Specifically, mice deficient in Bgn develop age-dependent osteopenia and have multiple metabolic defects in their bone marrow stromal cells, including increased apoptosis, reduced numbers of colony-forming units, and decreased collagen production (48). Therefore, the strategy of using mice deficient in one or more SLRPs has enabled the identification of early molecular events causing skeletal abnormalities that are dependent on SLRP function.

In view of the fact that the BGN gene maps to the X chromosome, patients with Turner syndrome (45 chromosomes, X0) have reduced biglycan expression and exhibit short stature, infertility, and early-onset osteoporosis. In contrast, patients with supernumerary sex chromosomes (e.g. Klinefelter syndrome) show increased biglycan expression and longer limbs (49). Thus, bone metabolism in biglycan-deficient mice could be gender-dependent. Indeed, in contrast to male mice, the bone tissue of female mice is less affected, suggesting a gender difference in biglycan skeletal function.

Biglycan can also control key signaling pathways regulating the osteogenic program, including the activity of the multifunctional cytokines TGF-β (50), BMP4 (22, 51), and Wnt (52) (Fig. 1, C–E). Consequently, biglycan can affect the number of available osteogenic precursors as well as their subsequent development, differentiation, and function in bone formation.

Phenotypic analysis of Dcn^−/−/Bgn^−/− double-null mice reveals that the pathological bone phenotype is more severe and appears evident earlier compared with Bgn-deficient mice. In addition to the gross skeletal phenotype appearance, Bgn and Dcn double deficiency results in a striking change in collagen fibril shape and organization to an extent that is greater than the additive effect. Therefore, the effects of Dcn/Bgn deficiency in bone are likely synergistic even though Dcn deficiency does not appear to affect bone phenotype by itself (47). This could be explained partly by the suggestion that the absence of biglycan and decorin prevents TGF-β from proper sequestration within the ECM. First, decorin and subsequently biglycan, asporin, and fibromodulin were found to bind TGF-β (21, 53), forming SLRP·TGF-β complexes, which are either eliminated from the tissue (via the circulation or by urinary excretion) or, in the presence of collagen I, are sequestered in the ECM, thus down-regulating TGF-β signaling (54–56). However, the interaction of SLRPs with TGF-β could also enhance the bioactivity of TGF-β, as seen in the case of decorin during the process of bone formation during remodeling (57) or muscle formation (58). Therefore, the direct binding of excess TGF-β to its receptors on bone marrow stromal cells could cause a “switch in fate” from growth to apoptosis and thus ultimately lead to decreased numbers of osteoprogenitor cells and subsequent reduced bone formation (50). Furthermore, biglycan promotes osteoblast differentiation through ERK phosphorylation and signal transduction through the transcription factor Runx2 (Fig. 1C) (59); alternatively, the biglycan protein core can bind to BMP2 and BMP2·ALK6 (activin receptor-like kinase 6), thereby sustaining the binding of BMP2·ALK6 (Fig. 1D). The BMP2·biglycan·ALK6 complex can phosphorylate Smad1/5/8 and thus facilitate their downstream signaling relevant to osteogenesis (60). Recently, biglycan was suggested to support Wnt signaling in vivo in a manner beneficial to bone formation (52). In this latter study, biglycan promoted binding of Wnt3a to bone matrix by a direct interaction that involved further interface with a key Wnt coreceptor in bone LRP6 (low-density lipoprotein receptor-related protein 6) (Fig. 1E).

Lumican is a major proteoglycan component of bone matrix. It is secreted by both differentiating and mature osteoblasts but not by proliferating pre-osteoblasts, and therefore, it can be used as a marker to distinguish proliferating pre-osteoblasts from differentiating osteoblasts (61). Keratocan expression is correlated with a more differentiated osteoblast phenotype, whereas histomorphometric analysis indicates that Kera^−/− mice have significantly decreased rates of bone formation and mineral apposition (62). Male epiphycan (Dspg3)-deficient mice also have significantly shorter femurs than wild-type mice at 9 months. Knee joints from Dspg3/Bgn double-null mice display increased matrix protein expression compared with wild-type mice. This enhanced expression also includes other SLRP members such as asporin, fibromodulin, and lumican, demonstrating compensatory mechanisms among different SLRP family members (63). The importance of SLRPs in bone homeostasis is further illuminated by the finding that individual members show species-specific and age-related changes that correlate with altered homeostasis of the aging skeleton (64). Another example of the importance of SLRPs in bone biology is offered by proline/arginine-rich end leucine-rich repeat protein, which can down-regulate osteoclastogenesis apparently by inhibiting NF-κΒ activity (65).

Roles in Degenerative Bone Diseases

SLRP biology is also closely linked to degenerative skeletal processes. Earlier studies have shown that targeted disruption of the Bgn gene leads to an osteoporosis-like phenotype in mice (46). Subsequently, it was proposed that the altered collagen phenotypes in mice deficient in one or more SLRPs demonstrate a cooperative, sequential, timely orchestrated action of the SLRPs that altogether shape the architecture and mechanical properties of the collagen matrix (66). The development of osteoporosis among other disease states by SLRP-deficient mice suggests that mutations in SLRPs may be part of undiagnosed predisposing genetic factors specific for the disease (66). On the other hand, biglycan deficiency was found to protect against increased trabecular bone turnover and bone loss in response to estrogen depletion, supporting the concept that biglycan may modulate both formation and resorption, ultimately influencing the bone turnover process (67).

In addition to normal bone turnover, the SLRPs have been shown to contribute to ectopic ossification (68) of soft tissues. Although both singly depleted biglycan and fibromodulin mice develop ectopic bone in tendon, the effect is exaggerated when the two SLRPs are deleted simultaneously, thus suggesting synergistic activities for these two SLRPs on this function (69). Recently, in vivo osteolysis experiments showed that LPS-induced osteolysis occurred more rapidly and extensively in Bgn-deficient mice compared with wild-type mice due to increased osteoclast differentiation and activity secondary to defective osteoblasts (70). Moreover, deregulation of matrix protein expression, including that of biglycan, was demonstrated in hypophosphatemia (Hyp) mice with osteomalacia (71). Considering that the depletion of SLRPs exacerbates degenerative diseases such as osteoporosis, osteoarthritis, and ectopic bone formation and taking into account an evermore aging population, rife with skeletal issues, one should consider novel therapies such as the use of SLRPs to ameliorate these pathological conditions.

Roles in Bone Tumor Progression

Malignant bone tumors are characterized by the presence of undifferentiated osteoprogenitors that proliferate and develop a mass of tumor tissue recognized as osteosarcoma. The expression and consequent function of SLRPs in primary bone tumors are known to differ compared with disease-free tissue. The absence of biglycan message observed in osteosarcoma samples and some osteosarcoma cell lines provides evidence for alterations in the extracellular matrix that result with non-mineralized osteoid produced by the osteosarcoma cells (72). Dissection of the sets of genes that control the behavior of Bgn-null pre-osteoblasts using oligonucleotide microarrays revealed that Bgn deficiency affects the genes that control inflammation, immune response, and growth of tumor cells (73). Indeed, biglycan affects osteoblastic tumor cell behavior, as it was recently suggested that osteosarcoma cells utilize Runx2 and the FGF-2/syndecan/heparan sulfate proteoglycan axis, a key effector of the osteogenic program, to regulate their migration in a manner dependent on biglycan expression (74). Furthermore, osteosarcoma markers of poor response to therapy (Huvos grade I/II response defines tumors with little or no response to chemotherapy) are predominantly gene products involved in microenvironmental remodeling and osteoclast differentiation, including Bgn (75). On the other hand, gene chip analysis has demonstrated that biglycan has the highest expression (out of 137 genes) in giant cell tumors of bone (76). Moreover, a series of chondrosarcomas, malignant tumors of cartilage epidemiologically established as the second most common primary malignant neoplasm of the skeleton in adults (77), were found to have up-regulated DCN transcript levels (78), whereas a human chondrosarcoma cell line was found to express higher amounts of BGN and not to express DCN (79).

Decorin had severalfold reduced expression in osteoblastic osteosarcoma patients compared with non-osteoblastic osteosarcoma patients, which could be correlated to a poorer prognosis of the first patient group (80). Immunohistochemistry using polyclonal and monoclonal antibodies against ECM molecules, including decorin, has proved to be a useful tool for the differential characterization of osteoid in a series of 20 osteosarcomas with different variants of osteoid formation (81). Importantly, several studies have shown that the histological subtype of osteosarcoma is a predictive factor for response to chemotherapy (82, 83) and correlates with a disease-free period (84, 85) and overall survival (86). Moreover, decorin was found to suppress murine osteosarcoma lung metastasis, which correlated to lower adhesion and motility capabilities of the decorin-expressing osteosarcoma cells (87). Collectively, these findings strongly correlate with the well established antitumor properties of decorin (88). Interestingly, the only exception to the established oncosuppression model is the case of the highly aggressive MG-63 human osteosarcoma cells, found to constitutively produce decorin and to be resistant to decorin-induced growth arrest (89).

The role of lumican in osteosarcoma pathogenesis has previously been reviewed (90). Indeed, the generation of LUM-deficient osteosarcoma cells suggested that LUM expression may be positively correlated with the differentiation and negatively correlated with the progression of osteosarcoma (40). Furthermore, a novel out-in signaling circuit in human osteosarcoma cells was described: secreted lumican was found to be an endogenous inhibitor of TGF-β2 activity, resulting in downstream effector modulation, including phospho-Smad2, integrin β1, and phosphorylated focal adhesion kinase to regulate osteosarcoma adhesion (91). On the other hand, fibromodulin expression was shown to be regulated by the BMP signaling pathway and thus possibly correlated to osteoblast origin cell transformation (92).

Potential Therapeutic Agents for Bone Diseases

As regulators of signaling molecules, SLRPs may hold promise for treatment and prevention of disease. The modulation of Bgn gene expression has been proposed to represent a mechanism to counteract mineralized bone loss under conditions of estrogen depletion (67). Trans-osseous application of low-intensity ultrasound at the tendon graft-bone healing interface stimulated endogenous expression of Bgn and collagen I, thereby enhancing tendon-bone healing (93). The above studies support utilization of biglycan therapy as a credible therapeutic venue to reduce or block degenerative bone diseases. Proof of principle for the possibility of such an application derives from a recent study showing that injections of biglycan into dystrophin-deficient mdx mice can repair numerous defects that mimic muscular dystrophy, including the improvement of muscle function (94). In this regard, it can be noted that exogenously applied recombinant biglycan could “rescue” impaired LRP6 signaling caused by a mutation in the extracellular domain of this Wnt receptor (54). Thus, the specific location and function of SLRPs outside the cell lend themselves to accessibility for effective and simple therapeutic intervention.

In the case of primary bone tumors (osteosarcomas), decorin-expressing osteosarcoma cells were found to have a decreased ability to generate pulmonary metastases in a mouse model, thus demonstrating that decorin has the therapeutic potential to reduce lung metastasis in osteosarcoma (87). One important aspect in considering the role of SLRPs in tumor growth and establishment is the role of the tumor microenvironment. Indeed, many SLRPs, including biglycan, decorin, and fibromodulin, are expressed in the stroma and, at least in the case of fibromodulin, appear to control its structure and integrity (95).

Concluding Remarks

In conclusion, this minireview has highlighted the important roles that some SLRPs have in bone physiology and disease. These small proteoglycans are established to specifically regulate both bone osteogenesis and remodeling as well as to participate in the progression of commonly debilitating degenerative bone diseases. Further progress in discerning the specific signaling pathways in skeletal development and homeostasis is needed. Because of SLRP participation in multiple degenerative, inflammatory, and neoplastic diseases, we expect that some of the SLRP-secreted gene products could become reliable biomarkers for bone diseases and perhaps become targets of novel therapeutic interventions. Finally, we should point out that some of the pharmacological therapeutics currently used in clinical settings such as corticosteroid and anti-inflammatory drugs have profound effects on SLRP synthesis and accumulation. These, in turn, could affect not only the structural composition of bone but also the myriad of bone-specific growth factors that ultimately affect bone remodeling and diseases.