Synovial joints develop through two main processes called joint specification and joint morphogenesis (reviewed in Pacifici et al., 2005). In the step of joint specification, skeletogenic MSCs expressing the Sox trio are directed to the articular fate under the control of TGF-beta receptor 2 (Spagnoli et al., 2007), canonical Wnt (e.g., Wnt4 and Wnt9a/14) (Hartmann and Tabin, 2001; Guo et al., 2004), and growth differentiation factor-5 (Gdf5) (Settle et al., 2003) signaling cascades, resulting in the formation of presumptive joint regions called interzones. Subsequently, a cavity forms in the interzone between the articulating skeletal elements driven by a not fully understood mechanism involving high-level synthesis of hyaluronan (Matsumoto et al., 2009), specific ECM composition (Pacifici et al., 1993; Choocheep et al., 2010), and skeletal movement (Kahn et al., 2009). During joint morphogenesis, specific articular cells differentiate and form the joint tissues. The continued expression of the Sox trio is pivotal for articular chondrocyte differentiation, while it seems that Wnt/beta-catenin signaling prevents chondrocyte differentiation at sites not destined for AC, such as the synovial lining and ligaments.
Integrins and Joint Morphogenesis
The function of integrin-mediated cell–cell or cell–matrix interactions in joint determination and morphogenesis, although anticipated (Pacifici et al., 2005), is poorly investigated and understood. Almost a decade ago, Garciadiego-Cázares et al. (2004) reported that α5 integrin is downregulated in the interzones of developing mouse fingers, suggesting a role for α5β integrin in patterning of the hands. Indeed, blocking integrins in E14.5 mouse forelimb organ culture by microinjecting anti-β1 and anti-α5 monoclonal antibodies or RGD peptides into the wrist region induced ectopic joint formation at the distal part of the radius and the ulna. A narrow gap region expressing interzonal markers, such as Gdf5 and Wnt9a/14, but not expressing the prehypertrophic marker Indian hedgehog (Ihh), had formed in between the proliferative and hypertrophic zones. In contrast, forced expression of human cDNAs coding for human α5 and β1 integrin subunits in embryonic chick legs had resulted in joint fusions of the phalanges, accompanied by the differentiation of prehypertrophic chondrocytes expressing Ihh. Consequently, the authors proposed that α5β1 integrin-RGD ligand (fibronectin) interactions are pivotal in cell fate determination during the development of the appendicular skeleton by dictating either the differentiation of interzonal cells or prehypertrophic chondrocytes on the basis of α5β1 expression level in chondrocytes. Although this hypothesis is clearly attractive, genetic experiments with conditional knockout mice did not support a mechanistic role for α5β1-mediated matrix attachments in joint morphogenesis. To date, no in vivo experiments have been performed to investigate the role of α5 integrins in skeletogenesis. However, neither β1fl/flCol2cre mice nor β1fl/flPrx1cre mice display ectopic joint formation, and in both models, Ihh is expressed at the normal level in the prehypertrophic zone (Aszodi et al., 2003; Raducanu et al., 2009). The lack of any obvious phenotype in joint formation in the absence of β1 integrins on skeletogenic MSCs, perichondrial cells, and chondrocytes implies that antibody perturbation might trigger non-specific and/or non-physiological responses (e.g., apoptosis) not seen upon genetic ablation.
Integrins and the AC
AC is a permanent, specialized type of hyaline cartilage that functions as a load bearing tissue and can resist shearing forces and friction. The AC, similar to the growth plate, is characterized by a high degree of anisotropy and is divided into four zones with distinct organization of the matrix and cells: superficial (or tangential), intermediate (or transitional), deep (or radial), and calcified (Poole et al., 2001). The superficial zone is the thin, outermost layer of the AC, which contains elongated cells and densely packed collagen fibrils arranged parallel to the surface. This zone has low aggrecan content but it is enriched in small proteoglycans, such as decorin and biglycan (Poole et al., 1996). The intermediate zone contains individual spherical chondrocytes and a relatively disorganized collagen network, whereas in the deep zone the cells tend to organize into columns with radially oriented collagen fibril bundles between them. The pericellular compartment of these zones has high collagen VI, decorin, and perlecan content, while aggrecan is more abundant in the territorial and interterritorial regions. The calcified zone is separated from the upper zones by the tidemark and its main function is to anchor the AC to the subchondral bone. The hypertrophic chondrocyte marker collagen X is expressed in this zone (Gannon et al., 1991; Hoyland et al., 1991; von der Mark et al., 1992), but unlike in the growth plate, the calcified matrix normally resists vascular invasion and resorption.
AC chondrocytes have a similar set of integrin heterodimers as growth plate chondrocytes with the most prominent expression of α1β1, α2β1, α10β1 (for collagens); α5β1, αvβ3, αvβ5 (for fibronectin), and α6β1 (for laminin) (Durr et al., 1993; Woods et al., 1994; Salter et al., 1995; Camper et al., 2001). In vitro studies revealed that human AC chondrocytes primarily use β1 integrins, including α5β1 and αvβ5 integrins, for adhesion to the cartilage matrix (Kurtis et al., 2003), whereas α5β1-fibronectin interaction was shown to promote survival of cultured AC chondrocytes (Pulai et al., 2002). Therefore it is not surprising that, analogous to the growth plate, β1 integrins are critical regulators of chondrocyte phenotype in the AC as well. β1fl/flPrx1cre mice display severe abnormalities of AC chondrocytes including rounding of the superficial zone cells, loss of columnar arrangement in the deep zone, reduced proliferation, increased cell death and bi-nucleation, and disrupted actin cytoskeleton (Raducanu et al., 2009). These observations extend the view that in vivo β1 integrins are pivotal for maintaining the normal shape, organization, proliferation, and survival of chondrocytes, independent of their location in the cartilaginous skeleton. Furthermore, β1 integrin-deficiency of AC chondrocytes leads to prominent ECM defects, including disorganized collagen II network in the interterritorial compartment and reduced deposition of collagen VI in the pericellular compartment, suggesting that β1 integrins are important organizers of the collagenous matrices. Importantly, the ablation of β1 integrins differentially affects chondrocyte maturation in the growth plate and the AC. While in β1fl/flCol2cre and β1fl/flPrx1cre mice the hypertrophic differentiation of growth plate and epiphyseal chondrocytes is delayed, the AC in the β1fl/flPrx1cre mice exhibits reduced ratio of the non-calcified and calcified zones and contains collagen X expressing chondrocytes above the tidemark, suggesting accelerated hypertrophic differentiation (Aszodi et al., 2003; Raducanu et al., 2009). This may imply that β1 integrins have a spatially restricted, tissue context- dependent role in chondrocyte maturation.
Interestingly, while no single α subunit-deficient mice have been reported with structural defects of the AC, the secreted melanoma inhibiting activity/cartilage-derived retionic acid sensitive protein (MIA/CD-RAP)-deficient mice partially recapitulate the AC abnormalities seen in the β1fl/flPrx1cre mice. MIA/CD-RAP is expressed in chondrocytes (Dietz and Sandell, 1996; Bosserhoff et al., 1997) and its absence in mice leads to mild ultrastructural abnormalities of the collagen fibrils in the developing growth plate cartilage (Moser et al., 2002). In 3 month old mice, MIA/CD-RAP-deficient AC shows disturbed columnar arrangement of the chondrocytes and increased height of the calcified zone (Schubert et al., 2010). Based on in vitro studies with cultured chondrocytes, it was proposed that MIA/CD-RAP physically binds to and inhibits α5β1 integrin, leading to reduced ERK signaling, stabilization of the chondrocyte phenotype, and delay of hypertrophy. This finding is consistent with a recent report demonstrating correlation between ERK activation and hypertrophic differentiation of human AC chondrocytes (Prasadam et al., 2010). Furthermore, an antibody-activating α5β1 integrin/ERK signaling (Forsyth et al., 2002) was shown to abolish the negative effect of MIA/CD-RAP on the expression of the osteogenic marker osteocalcin in cultured AC chondrocytes (Schubert et al., 2010).
Taken together, in vitro and loss-of-function genetic studies in mice strongly suggest that integrins are required for proper differentiation and spatial organization of AC chondrocytes. Since the ablation of both β1 integrin and the α5β1 integrin-inhibitory MIA/CD-RAP results in a similar phenotype, the exact nature of integrin-mediated signaling pathways on articular chondrocyte hypertrophy remains to be elucidated.
Integrins and Osteoarthritis
AC is highly prone to injury and pathological degeneration. Once damaged, the AC is unable to initiate an efficient regenerative process, owing to the loss of mitotic activity of adult AC chondrocytes, decreased responsiveness to anabolic growth factors, and synthesis of less functional matrix molecules. Osteoarthritis (OA) is a common degenerative disorder of the synovial joints that not only affects the AC but also involves changes in subchondral bone, synovium, ligaments, and muscle. OA is a complex disorder influenced by both environmental and genetic factors. Although the precise etiology is largely unknown, it is generally accepted that uncontrolled metabolism of skeletal tissues is critical for the pathophysiology of this disabling condition. In primary OA, the normal turnover of ECM molecules produced by chondrocytes is disturbed, and the equilibrium between anabolic and catabolic processes is shifted towards proteolytic activities, which eventually results in progressive, age-dependent AC destruction (Goldring and Goldring, 2007). In addition, congenital abnormalities of the joints, as a typical result of impaired development of endochondral bones, are often associated with early onset degeneration and loss of the AC (secondary OA). Physiological ECM remodeling of the AC occurs in a spatially and temporally controlled fashion, and involves the activities of both proteinases and proteinase inhibitors, which are tightly regulated at multiple levels. The regulatory mechanisms are only partially understood and include, in addition to growth factor and cytokine signaling pathways, chemical and mechanical signals modulated by matrix–matrix and cell–matrix interaction (Knudson and Loeser, 2002). There is a growing consensus that changes of ECM composition, e.g., by proteolytic degradation of matrix constituents; or alterations of the biomechanical microenvironment of chondrocytes caused by chronic stress or injury, significantly increase the risk of OA through the perturbation of integrin-mediated signaling.
The strongest evidence for the participation of integrins in OA has emerged from experiments studying integrin expression and ECM fragments-induced signaling processes. In osteoarthritic monkey AC, increased immunostaining of α1, α3, and α5β1 integrins was reported, compared with normal cartilage (Loeser et al., 1995). In human OA samples, the expression of β1 integrin was inversely correlated with the severity of the lesions, suggesting that β1 integrin-mediated-ECM adhesion is decreased with OA progression (Lapadula et al., 1998). In contrast, osteoarthritic human femoral head cartilage expressed the integrin subunits β2, α2, and α4, which were not observed in normal AC (Ostergaard et al., 1998). Subsequently, numerous reports have shown that fibronectin (FN) and collagen II degradation fragments are present in synovial fluid and cartilage of OA patients as a result of increased catabolic activity, and these fragments can further mediate joint destruction through the activation of integrin signaling (reviewed in Yasuda, 2006, Sofat, 2009). Treatments of cultured rabbit synovial fibroblasts with the central, 120 kDa cell-binding FN fragment (FN-f) carrying the RGD sequence or ligation of α5β1 integrin with a monoclonal antibody were shown to stimulate MMP-1 and MMP-3 expression (Werb et al., 1989). Similarly, the central FN-f and α5β1/α2β1 activating antibodies were shown to upregulate MMP-13 synthesis in cultured human AC chondrocytes (Forsyth et al., 2002; Loeser et al., 2003). Mechanistically, the stimulation of α5β1 integrin with ligation antibodies or FN-f activates protein kinase C (PKC)-delta, which in turn phosphorylates the proline-rich tyrosine kinase-2 (PYK2), resulting in increased MAPK (Erk-1/2, p38, and JNK) signaling and increased phosphorylation of c-jun and NF-kappa B (Loeser et al., 2003; Pulai et al., 2005; Ding et al., 2008). In addition to central FN-f, the amino-terminal 29 kDa and the gelatin-binding 50 kDa FN-fs are also chondrolytic and likely interfere with the native FN/α5β1 signaling pathway (Homandberg and Hui, 1994; Homandberg et al., 2002a, b). The physiological relevance of Fn-fs in cartilage destruction was evidenced by studies demonstrating that injection of Fn-fs into knee joint dramatically increases proteoglycan loss of rabbit AC (Homandberg et al., 1993). The exact mechanism by which ECM degradation fragments accelerate cartilage catabolism is unclear. The most popular explanation supposes that FN and collagen degradation products disturb or even disrupt normal integrin clustering induced by native FN or collagen II, which in turn alters physiological outside-in signaling and cartilage metabolisms (Peters et al., 2002). However, beside degradation fragments, collagen itself is able to induce MMP expression upon binding to α1β1 integrin. Collagen I is upregulated in OA cartilage (Adam and Deyl, 1983; Miosge et al., 2004) and in the chondrogenic cell line MC615, and it stimulates the expression of MMP-13 via the activation of ERK and JNK MAP kinases. In the presence of α1 or β1 blocking antibodies the induction of MMP-13 expression was inhibited (Ronziere et al., 2005). Furthermore, a recent report has suggested that collagen II fibrils modified by the lipid peroxidation end-product 4-hydroxynonenal, which is upregulated in synovial fluid of OA patients, induce cell death and catabolic and inflammatory responses of human AC chondrocytes, presumably through perturbation of α1β1 signaling (El-Bikai et al., 2010).
In AC, physiological mechanical loading leads to anabolic changes that help to maintain tissue integrity, while non-physiological mechanical stimuli (e.g., overloading and injury) are associated with cartilage damage and predispose to OA. Chondrocytes that are exposed to mechanical forces transmit the signals via various mechanotransducers, including stretch activated ion channels, receptor tyrosine kinases, the hyaluronan receptor CD44, and integrins (Millward-Sadler and Salter, 2004; Bader et al., 2011; Vincent, 2013). Under physiological loading, integrins (e.g., α5β1) are activated on the surface of isolated chondrocytes, leading to recruitment of adaptor proteins in focal adhesion complexes and to lateral interactions with growth factor receptors and ion channels (Millward-Sadler et al., 2004). Induction of the integrin signaling pathway may lead to the secretion of interleukin-4 (IL-4), which via autocrine/paracrine signaling events, blocks catabolic processes and enhances GAG and proteoglycan synthesis (Millward-Sadler et al., 1999; Holledge et al., 2008). The application of dynamic cyclic compression on human chondrocytes in the presence of TGFβ3 also enhanced proliferation and proteoglycan synthesis, which was reversed by adding the α5β1 ligand GRGDSP to the system (Chowdhury et al., 2004). Mechanotransduction via α5β1 might be regulated by the α5 integrin-associated protein CD47, since a function-blocking anti-CD47 antibody inhibited membrane hyperpolarization, tyrosine phosphorylation, and the elevation of aggrecan mRNA expression induced by mechanical stimulation (Orazizadeh et al., 2008). In contrast to this protective role, non-physiological mechanical loading disrupts the actin cytoskeleton via α5β1-mediated signaling that activates the MAP kinases and NF-kappa B. These, in turn, result in elevation of nitric oxide, prostaglandin E2, reactive oxygen species, proteases, and cytokines, which mediate AC degradation (Bader et al., 2011).
Although these expression studies and in vitro experiments highlight the importance of integrin signaling in OA and suggest that inhibition of integrin pathways might be a potential approach to ameliorate ECM and ECM fragment-mediated AC destruction, the in vivo role of integrins in OA pathogenesis has just begun to be elucidated. Most mutant lines lacking a particular α subunit develop an apparently normal AC without reported abnormalities. The collagen-binding α1β1 integrin normally is expressed at low levels in the deep zone of healthy mouse AC but it is significantly upregulated in the middle and upper zones of early osteoarthritic cartilage, suggesting that this integrin heterodimer supports cartilage remodeling (Zemmyo et al., 2003). Knee joints of α1-null mice display precocious proteoglycan loss, cartilage erosion associated with increased MMP-2 and MMP-3 expression, and synovial hyperplasia (Zemmyo et al., 2003). Accelerated development of knee OA was also observed in mice lacking the membrane-anchored disintegrin and metalloproteinase ADAM15 (Bohm et al., 2005). ADAM15 carries a RGD motif and has multiple pericellular functions, including the modulation of outside-in signaling in chondrocyte-matrix interactions (Bohm et al., 2009), which in turn enhances chondrocyte adhesion to collagen II and promotes AC homeostasis. Interestingly, β1fl/fl-Prx1cre+ mice, despite the severe chondrodysplasia and efficient deletion of β1 integrins in AC chondrocytes, showed no significant differences in erosion of the knee AC, in the expression of matrix-degrading proteases, or in the exposure of aggrecan and collagen II cleavage neoepitopes, compared with controls (Raducanu et al., 2009). In contrast to the previous in vitro experiments, no evidence was found for disturbed activation of MAP kinases in mutant AC and in hip explants upon stimulation with a 40 kDA cell-binding FN fragment. These results suggest that either β1 integrins are not required for ECM–ECM fragment induced MAPK activation per se, or, and this is the most likely scenario, the lack of β1 integrins is efficiently compensated via signaling through β3/β5 integrins.
AC has a poor intrinsic capacity to heal owing to limited synthetic and proliferative potential of AC chondrocytes; low efficiency of AC resident chondrogenic progenitors to induce rapid and successful regeneration; and the lack of vascular system to deliver MSCs into the damaged tissue. Current orthopedic therapeutic strategies include marrow stimulation-based, osteochondral transfer, and cell-based techniques (Richter, 2009), with various advantages and disadvantages of each. Subchondral bone drilling stimulates the recruitment, proliferation, and chondrogenic differentiation of bone marrow-derived MSCs, but it frequently produces fibrocartilage, which has a different biochemical composition and insufficient biomechanical properties, compared to native hyaline cartilage. Autologous chondrocyte implantation (ACI) and the more recent matrix assisted ACI (MACI) have provided encouraging clinical results to heal cartilage defects; however, these techniques are hampered by the low proliferation rate of isolated chondrocytes in culture and their high de-differentiation potential to fibroblasts during the expansion period. MSCs isolated from various sources (e.g., bone marrow, synovial membrane, adipose tissue) have sufficient proliferative activity in vitro but require special culture conditions to differentiate into chondrocytes. Inducing and/or stabilizing the chondrogenic phenotype is a crucial requirement of all regenerative therapies, and the importance of integrins in this process has been increasingly recognized.
MSCs differentiation into different lineages (chondrogenic, osteogenic, adipogenic) in vitro can be modulated via numerous factors, including growth factors, cell–matrix interactions, 3D microenvironment, cytoskeletal tension, or mechanical stimulation (McBeath et al., 2004; Engler et al., 2006; Woods et al., 2007; Kelly and Jacobs, 2010). Collagen II, for example, enhances chondrogenesis of both bone marrow-derived MSCs (BMSC) and adipose-derived MSCs (ASC) in hydrogels (Bosnakovski et al., 2006; Lu et al., 2010) by inducing rounded cell shape through β1 integrin-mediated RhoA/Rock signaling (Lu et al., 2010). Co-culturing BMSCs and collagen II fibrils in 3D led to remodeling of the fibers, differentiation of spherical chondrocytes, and synthesis of cartilaginous markers on β1 and α2 integrin-dependent manner (Chang et al., 2007). In contrast, interaction with RGD peptides inhibits chondrogenesis of BMSCs by promoting spreading through αvβ3 integrin and actin cytoskeletal reorganization (Connelly et al., 2007, 2008). Stem cell commitment can be further regulated via ECM remodeling. The membrane-bound MT1-MMP (MMP-14), a pericellular collagenase, modulates cell shape, which in turn activates a signaling pathway involving β1 integrin, Rho GTPase activity, and the YAP/TAZ transcriptional coactivators (Tang et al., 2013). Using MT1-MMP-deficient MSCs and mice lacking MT1-MMP in skeletogenic MSCs (Mmp14fl/fl/Dermo1-cre), it was shown that the absence of this protease directs MSC fate towards chondrogenesis and adipogenesis at the expense of osteogenesis both in vitro and in vivo. Stiffness of the hydrogels and external mechanical loading (e.g., cyclic compression and hydrostatic pressure) also influence MSC chondrogenesis (Huang et al., 2004; Pelaez et al., 2009; Meyer et al., 2011), which involves integrin-mediated cell-matrix interactions (Steward et al., 2012, 2013). Recently, an α10 integrin expressing subpopulation of monolayer expanded human BMSCs with high chondrogenic potential in pellet culture was identified, suggesting that α10β1 can be a unique biomarker for quality assurance of MSCs used for cartilage repair (Varas et al., 2007). Furthermore, α10 expression was significantly downregulated during extensive monolayer culture of BMSCs, which was reversible by FGF-2 treatment.
Further in vitro studies have demonstrated the prospect of the modulation of integrin signaling in AC repair. The activation of FAK is increased upon exogenous stimulation of alginate beads with collagen II, whereas FAK knockdown reduces chondrocyte re-differentiation in the same culture system (Kim and Lee, 2009). As discussed earlier, MIA/CD-RAP induces chondrogenic differentiation via abolishing α5 integrin activity and inhibiting the downstream ERK signaling; and stimulates cartilage synthesis of chondrocytes obtained from patients suffering from OA (Tscheudschilsuren et al., 2006; Schubert et al., 2010). Importantly, MIA/CDRAP-deficient mice exhibit increased regeneration potential of the AC in a surgically induced OA model because enhanced proliferation of the chondrocytes (Schmid et al., 2010). In contrast, deficiency of tenascin C, a ligand of some β1 and β3 integrins, delays the repair of full thickness cartilage defects in mice (Okamura et al., 2010). Finally, a recent report has shown that magnesium enhances the adhesion and hyaline cartilage differentiation of human synovial MSCs at the sites of osteochondral plugs in the rabbit knee through activation of β1 integrins (Shimaya et al., 2010).