Hypertrophic differentiation of chondrocytes in osteoarthritis: the developmental aspect of degenerative joint disorders

Osteoarthritis is characterized by a progressive degradation of articular cartilage leading to loss of joint function. The molecular mechanisms regulating pathogenesis and progression of osteoarthritis are poorly understood. Remarkably, some characteristics of this joint disease resemble chondrocyte differentiation processes during skeletal development by endochondral ossification. In healthy articular cartilage, chondrocytes resist proliferation and terminal differentiation. By contrast, chondrocytes in diseased cartilage progressively proliferate and develop hypertrophy. Moreover, vascularization and focal calcification of joint cartilage are initiated. Signaling molecules that regulate chondrocyte activities in both growth cartilage and permanent articular cartilage during osteoarthritis are thus interesting targets for disease-modifying osteoarthritis therapies.


Introduction
Osteoarthritis (OA) is the most common joint disorder in western populations. Its incidence increases with age, and thus this degenerative disease is a major problem in ageing populations. Th e disease is characterized by a progressive degradation of articular cartilage leading to loss of joint mobility and function accompanied by chronic pain. On the biochemical level, OA is characterized by uncontrolled production of matrix-degrading enzymes, including aggrecanases (a disintegrin and metallo protease with trombospondine motifs (ADAMTSs)) and matrix metalloproteinases (MMPs), which result in the destruc tion of cartilage matrix [1]. Other hallmarks of the disease are new bone formation at the joint margins (osteophytes), limited infl ammation (synovitis), and changes in subchondral bone structure (sclerosis). Th e molecular mechanisms regulating pathogenesis and progression of OA, however, are only poorly understood, and no proven disease-modifying therapy is currently available. Remarkably, some characteristics of OA -that is, articular chondrocyte proliferation, the expression of hypertrophy markers (for example, MMP-13 and collagen X), remodeling of the cartilage matrix by proteases, vascularization and focal calcification of joint cartilage with calcium hydroxyapatite crystals -resemble chon dro cyte diff erentiation processes during skeletal develop ment by endochondral ossifi cation (EO). Signal ing molecules regulating chondrocyte activities in growth cartilage may thus also be involved in OA pathogenesis.
In the present review, current concepts for the control of late chondrocyte diff erentiation in EO will be discussed in the light of analogous events observed during the development of OA. Th is knowledge is essential for the successful development of future therapeutic strategies.

Endochondral ossifi cation
EO is important for development, growth, and repair of long bones. EO is initiated by the formation of cartilage templates of future bones, built by mesenchymal progenitor cells, which condensate and diff erentiate into chondrocytes. Within these bone anlagen, the diff er entiated cartilage cells then transit through a temporospatial cascade of late diff erentiation events that sequentially include proliferation and several steps of maturation, culminating in chondrocyte hypertrophy. After invasion of blood vessels from the subchondral bone, the majority of hypertrophic cells undergo apoptosis and the cartilage template is remodeled into trabecular bone [2]. Proliferation of chondrocytes, hypertrophic diff erentiation of chondrocytes, remodeling and mineralization of the extracellular matrix (ECM), invasion of blood vessels and apoptotic death of chondrocytes correspondingly occur during OA.
Each phase of EO is accompanied by a change in cell shape or cell arrangement [3,4] (Figure 1) and the expression of a specifi c protein repertoire. Collagen I, besides collagens III and V, is the major fi brillar component of undiff erentiated mesenchymal progenitor cells [5]. After diff erentiation into chondrocytes, the cells cease to produce collagens I, III, and V but start to express typical cartilage components, including collagens II, IX, and XI and the proteoglycan aggrecan [6]. During this diff erentiation stage these so-called resting chondrocytes are small, uniform and characterized by low proliferation rates. Th ese cells occur singly or in pairs, and in the resting zone the ECM takes more space than the cells. In the adjacent proliferative stage, the chondrocytes divide several times and the fl at cells arrange into longitudinal columns. Th e expression repertoire now includes collagen VI [7] and matrilin 1 [8] in addition to the collagens II, IX and XI and aggrecan. During prehypertrophy, Indian hedgehog (Ihh) is expressed [9]. Further diff erentiation into hyper trophic chondrocytes induces the production of collagen X. Hypertrophic chondrocytes also reduce, or even terminate, their production of collagens II, IX, and XI, and express MMP-13, alkaline phosphatase, vascular endothelial growth factor (VEGF), osteopontin, and the transcription factor Runx2 [10]. Collagen X, MMP-13, and alkaline phosphatase are well-established markers for the overt hypertrophic stage of late chondrocyte diff erentiation.

Regulation of chondrocyte diff erentiation in growth cartilage
Chondrocyte diff erentiation in growth cartilage is subject to positive and negative control elements that interact within a signaling network to regulate the rate and progression of the process. EO is controlled by locally acting autocrine signals derived from chondrocytes themselves or by paracrine signals derived from cells of surrounding tissues (for example, the perichondrium or subchondral blood vessels). Th e interaction of chondrocytes with their surrounding matrix via cell surface receptors is also thought to play a key role in the regulation of survival, proliferation and maturation of cartilage cells. Many stationary and diff usible regulators of chondrocyte diff erentiation as well as their cell surface receptors are proteins. Proteinases are thus not merely destructive eff ectors of ECM degradation but also intervene in regulatory networks, both by eliminating control elements (for example, endoplasmic reticulum protein 57 (ERp57) [11]) and by converting precursors into active agents (for example, transforming growth factor beta (TGFβ) [12]). In addition, proteinases modulate mediator activities by direct cleavage or by release from ECM stores (for example, VEGF [13]). Most signaling events culminate at the level of gene expression; thus transcription factors are also essential regulatory elements [10]. Several positive and negative feedback mechanisms exist, however, which complicate the signaling network.

Locally produced, secreted growth factors
Several locally produced factors -such as bone morphogenetic proteins (BMPs), fi broblast growth factors (FGFs), TGFβ, Wnts, Ihh, parathyroid hormone-related peptide and retinoids -are so far known to infl uence EO. Th e actions of each of these growth factors during EO are briefl y summarized below.
BMP signaling initiates chondroprogenitor cell diff eren tiation, but in later EO stages induces chondrocyte proliferation and inhibits hypertrophic diff erentiation via Smad transcription factors [14]. Proteins of the FGF family, however, antagonize the BMPs. FGF-2 inhibits longitudinal bone growth by three mechanisms: decreased proliferation of growth plate chondrocytes, decreased cellular hypertrophy, and, at high concentrations, decreased cartilage matrix production. Th ese eff ects may explain the impaired growth seen in patients with achondroplasia and related skeletal dysplasias [15]. FGF-2 inhibits chondrocyte hypertrophy in synergy with TGFβ 2 [16], while TGFβ alone inhibits chondrocyte proliferation, hypertrophy and mineralization [17]. In suspension cultures of chick sternum chondrocytes, TGFβ even initiates phenotypic changes of dediff erentiation [18]. On the other hand, TGFβ 1 has also been shown to increase alkaline phosphatase activity and to stimulate proliferation in rat costochondral cartilage cells through protein kinase C and protein kinase A signaling [19], suggesting a variability of TGFβ eff ects depending on the species, the diff erentiation status of the receiving cells and the TGFβ concentration.
Members of the Wnt family are involved in diff erent stages of EO. During mesenchymal condensation, Wnt signaling favors osteoblastic diff erentiation but prevents chondrogenic diff erentiation; whereas at later stages, canonical Wnt/β-catenin signaling is indispensable for chondrocyte maturation. Wnt/β-catenin signaling acts as a positive regulator of chondrocyte hypertrophy and subsequent ossifi cation [20]. Retroviral overexpression of Wnt9a (formerly known as Wnt14), one of the 19 ligands of the Wnt-signaling pathway, in chicken embryo limb buds results in a blockage of chondrogenic diff erentiation of the infected prechondrogenic region [21,22].
Ihh is expressed in the prehypertrophic chondrocytes of cartilage elements, where it regulates the rate of hypertrophic diff erentiation. In a feedback loop of paracrine control, perichondrial cells induced by the chondrocytederived Ihh produce parathyroid hormone-related peptide, which delays progression of late diff erentiation at late proliferative stages [9]. Additionally, proteins of the hedgehog family can also accelerate hypertrophic chondrocyte diff erentiation without involvement of parathyroid hormone-related peptide in vitro and in vivo [23], suggesting a direct eff ect of hedgehog proteins, possibly depending on the maturation stage of the receiving cell.
Th e vitamin A derivative retinoic acid positively regulates hypertrophic chondrocyte diff erentiation and matrix mineralization [24].

Hormones
In addition to locally produced growth factors, systemic hormones -such as growth hormone (GH), insulin-like growth factors (IGFs), thyroid hormone, androgen, estrogen and glucocorticoids -tightly regulate longi tudinal bone growth. Local and systemic agents control the rate and extent of chondrocyte proliferation and diff erentiation at several checkpoints. Th is endocrine control enables longitudinal bone growth in healthy individuals and leads to increased growth rates and subsequent growth plate closure around puberty. GH and IGFs are potent stimulators of longitudinal bone growth. Both factors stimulate proliferation of resting zone chondrocytes and initiate chondrocyte hypertrophy [25]. Some eff ects of GH are likely to be mediated by IGF-I, with locally produced IGF-I seeming more important than systemic IGF-I [26].
Th yroid hormone is also indispensable in EO. Hypothyroidism slows down longitudinal bone growth, whereas hyperthyroidism accelerates the process. In vitro and in vivo studies have confi rmed that thyroid hormone regulates the transition between cell proliferation and terminal diff erentiation in the growth plate; specifi cally, the maturation of chondrocytes into hypertrophic cells. Administration of thyroid hormone dose-responsively increases synthesis of type X collagen mRNA and protein, alkaline phosphatase activity, and cellular hypertrophy, all markers of the terminally diff erentiated phenotype of the growth plate chondrocyte [27].
Sex steroids are essential during the pubertal growth spurt and epiphysial fusion. Androgen stimulates chondro cyte proliferation and matrix production, and thereby contributes to the increased long bone growth during the pubertal growth spurt. Estrogen aff ects growth plate cartilage through systemic as well as direct eff ects. On the one hand, estrogen regulates the GH/IGF-I axis, leading to decreased longitudinal growth and closure of the growth plate [28]; but estrogen also interacts with its receptors α and β within the growth plate, mediating direct eff ects [29].
Th e role of vitamin D signaling during bone development is well known because vitamin D defi ciency leads to bone-softening diseases, such as rickets in children and osteomalacia in adults. Th ese abnormalities result from decreased apoptosis of hypertrophic chondrocytes, widening of hypertrophic zones, and impaired bone mineralization. Some of the vitamin D eff ects are indirect through vitamin D actions on intestinal calcium and phosphate uptake. 24,25(OH) 2 vitamin D 3 , however, directly reduces chondrocyte diff erentiation in resting zone chondrocytes and stimulates late diff erentiation, while 1,25(OH) 2 vitamin D 3 directly decreases proliferation and inhibits hypertrophic diff erentiation of proliferative cells through binding to a membrane-associated rapid-response steroid receptor (ERp57) on growth plate chondrocytes [11,30].

Extracellular matrix molecules
An intact fi brillar periphery is a prerequisite for normal cellular architecture of the growth plate, with collagen IX being particularly important for proliferation and maturation of chondrocytes. Newborn mice lacking collagen IX develop abnormal areas with strongly reduced cell numbers within the epiphysis of long bones. In addition, a disturbed columnar arrangement of chondro cytes was detectable, resulting in shorter and broader long bones especially in newborn collagen IX-defi cient mice [31]. Th e importance of cell-matrix interactions also was demonstrated in mice defi cient in receptor proteins on chondrocytes that interact with ECM molecules. Integrins α 1 β 1 , α 2 β 1 , and α 10 β 1 are the major collagen receptors in cartilage. Th eir role in the spatial arrangement of growth plate chondrocytes and in outside-in-signaling has been intensively studied. Integrins, and their downstream signals integrin-linked kinase and the Rho GTPase Rac1, seem to act in a common pathway regulating cartilage development and disturbances in outside-in-signaling from the ECM to the cytoskeleton contribute to severe skeletal phenotypes [32][33][34].
Integrins are not the only ECM receptors in cartilage. Th e discoidin domain receptors (DDRs) are members of a subfamily of tyrosine kinase receptors that are activated by a number of diff erent collagens, amongst others by collagens II and X. DDRs regulate cell proliferation, adhesion and motility, and control remodeling of the ECM by infl uencing the expression and activity of MMPs; for example, MMP-13 [35]. Mice lacking DDR2 exhibit dwarfi sm due to decreased proliferation of growth plate chondrocytes [36].

Proteases
MMPs are members of a family of zinc-dependent proteolytic enzymes. Several MMPs are expressed in bone and cartilage at high levels and are essential for normal EO. MMPs are directly involved in the degradation of proteins such as collagens and proteoglycans necessary for remodeling of the cartilaginous template during EO. Of further interest are the ECM degradation products, called matrikines, which are involved in the induction of higher concentrations or additional catabolic enzymes, amplifying the ECM remodel ing. In addition to ECM degradation, however, several proteases are involved in the recruitment of cells into the growth plate through activation of recruitment factors (for example, VEGF) or infl uencing their bioavailability within the matrix [13].
Bone phenotypes are detectable in several mouse strains with MMP defi ciencies. Although hypertrophic chondrocytes of MMP-9-defi cient animals develop normally, apoptosis, vascularization, and ossifi cation are delayed, resulting in progressive lengthening of the growth plate [37]. Wu and colleagues observed that MMP-13 activity is required for chondrocyte diff erentiation associated with matrix mineralization [38]. Studies on mice with compound inactivation of the MMP-9 and MMP-13 genes reveal that both proteases act in a synergistic manner. Th e double-null mice display severely impaired endochondral bone formation, character ized by diminished ECM remodeling, prolonged chondrocyte survival, delayed vascular recruitment and defective trabecular bone formation, resulting in drastically shortened bones [39]. MT1-MMP (MMP-14) defi ciency causes a delay in the formation of the fi rst and second ossifi cation centers, a disorganized proliferation zone with reduced proliferation of chondrocytes, and an expanded zone of hypertrophic chondrocytes [40]. In addition the cysteine proteinases, especially the cathepsins, have also been implicated in several proteolytic scenarios during development, growth, remodeling, and aging. Th roughout endochondral ossifi cation, cathepsins B, H, K, L, and S were detected immunohistochemically in growth plates of rats and humans [41] and are thought to be involved in the proteolysis of several ECM components. We could show that cysteine proteinases mediate the shedding of 1,25(OH) 2 vitamin D 3 membraneassociated rapid-response steroid receptor (endoplasmic reticulum protein 57), trigger chondrocyte size expansion and trigger expression of collagen X, alkaline phosphatase, and MMP-13 as markers for overt hypertrophy [11].

Transcription factors
Gene expression during distinct chondrocyte maturation phases within the epiphysial cartilage or growth plates is controlled by transcription factors translating the environmental signals into regulated gene expression. Th e two main transcriptional regulators of chondrogenesis and hypertrophic diff erentiation -Sox9 and Runx2 -should be addressed.
Sox9 was characterized as the master gene of chondrogenesis that regulates proliferation and diff erentiation of nonhypertr ophic chondrocytes. Along with Sox5 and Sox6, Sox9 regulates the expression of aggrecan, and the α 1 chains of collagen II and collagen XI [42]. In addition, Sox9 acts as a negative regulator of chondrocyte hypertrophy, cartilage vascularization, and bone marrow formation [43].
Runx2 and its relative Runx3 are central positive regulators of the transition from proliferating to hypertrophic chondrocytes. Runx2/3 double-defi cient mice were shown to lack hypertrophic chondrocytes anywhere in the skeleton [44]. In addition, Runx2-defi cient mice lack upregulation of VEGF in hypertrophic chondrocytes and thus cartilage angiogenesis, suggesting that VEGF expression during bone development is controlled by the transcription factor Runx2 [45].
Another important transcription factor in bone develop ment, detected recently, is CCAAT/enhancerbinding protein beta (C/EBPβ). C/EBPβ defi ciency in mice was shown to cause dwarfi sm with elongated proliferative zones and delayed chondrocyte hypertrophy in growth cartilage [46]. Since growth arrest and DNA damage (GADD)45β -/animals [47] display similar phenotypes to the C/EBPβ knockout mice [46], the molecular interplay of GADD45β and C/ERBβ was analyzed in further detail. Various experiments indicate that GADD45β enhances C/ERBβ transactivation of the collagen 10a1 promoter and therefore is an upstream modulator of C/EBPβ [48].

Chondrocyte diff erentiation processes in articular cartilage
Articular cartilage is formed for life. Articular chondrocytes therefore display only moderate metabolic activity under normal conditions, primarily to maintain their surrounding ECM comprising collagens (collagens II, VI, IX and XI), proteoglycans (aggrecan, decorin, biglycan and fi bromodulin) and further noncollagenous matrix proteins. Under nondiseased conditions, the cells remain in a resting state and refrain from proliferation or ter minal diff erentiation. In a diseased state, however, some articular chondrocytes lose their diff erentiated phenotype; they enter an EO-like cascade of proliferation [49] and hypertrophic diff erentiation, accompanied by marker expression for the overt hypertrophic diff eren tiation stage, such as alkaline phosphatase [50], collagen X [51], and MMP-13 [52], with subsequent apoptotic death [53] and mineralization of the diseased cartilage [54] (Figure  2).
OA is considered a multifactorial disease; however, the scenario that OA is, at least in part, based on illegitimate hypertrophic diff erentiation should be taken into account. Diff erentiation of chondrocytes leads to an enhanced metabolic activity of articular chondrocytes, a change in the expression of ECM molecules, and an altered pattern of proteases. Altogether, this diff erentiation triggers a disturbed cartilage homeostasis favoring degenerative changes. Several signaling factors involved in chondrocyte proliferation and diff erentiation during endochondral ossifi cation were also shown to play a regulative role in OA cartilage, but not under nondiseased conditions. In all cases in which this signaling initiates the modulation of ECM or the expression or activation of proteases (for example, MMP-13 or aggrecanases), diff erentiation changes should be considered a potential driver of OA. A number of examples illustrating the analogy of signaling events in bone development by EO and cartilage degeneration in OA are given below. Th e reader should, however, keep in mind that most of the factors reviewed here were analyzed in spontaneous, transgenic or surgically induced mouse models of OA but not in large animals, which occasionally better refl ect human OA pathophysiology.

Growth factor signaling in osteoarthritis
Chondrocyte diff erentiation and matrix remodeling in osteoarthritic cartilage is regulated by BMPs, FGF-2, TGF-β, Wnts, hedgehogs and retinoids -all of which are also involved in the regulation of EO.
Although BMP-2 has potent anabolic actions, BMP activity in chondrosarcoma cells and in murine cartilage was shown to induce OA-like changes by stimulation of MMP-13 [55], directly favoring cartilage loss. FGF-2, however, displays benefi cial eff ects on articular cartilage homeostasis. Chia and colleagues observed that FGF-2defi cient mice had increased OA development with age as compared with wild-type mice. Th is is due to an increased expression of ADAMTS-5, the key murine aggrecanase [56]. Th e contrary role of BMP and FGF signaling pathways in OA was also described recently in respect of extracellular heparan sulfatases Sulf-1 and Sulf-2, which are found overexpressed in OA cartilage. Sulfs simul taneously enhance BMP signaling via Smad1/5 phos phorylation but inhibit FGF signaling via ERK1/2 phosphory lation, and thereby maintain cartilage homeostasis and favor cartilage repair [57].
Lack of TGFβ signaling results in OA-like changes with terminal diff erentiation of chondrocytes. As shown in genetically modifi ed mice, TGFβ mediates this eff ect by binding to the ALK5 type-I TGFβ receptor and subsequent activation of the Smad2/3 intracellular signaling route [17,58]. Notably, TGFβ supplementation can enhance cartilage repair and therefore was thought to serve as a potential therapeutic tool. Conversely, supplemen tation with TGFβ provides problems in noncartilaginous tissues of the joint and results in fi brosis and osteophyte formation in a murine model of OA [59]. Moreover, Van der Kraan and colleagues recently suggested a dual role for TGFβ in articular mouse cartilage because not only signaling via ALK5 (Smad2/3) but also via ALK1 (Smad1/5/8) can be initiated by TGFβ. Importantly, only signaling via ALK1, but not via ALK5, stimulates MMP-13 expression and thereby collagen degradation [60].
Recent genetic data of Caucasian test persons linked a polymorphism in the FrzB gene, encoding for a Wnt binding protein, to the development of OA, suggesting that abnormal Wnt signaling also contributes to OA [61].
Blom and colleagues found that β-catenin, along with a panel of other Wnt/Fz-related genes, was upregulated in cartilage and synovium during experimental OA in mice. Th e authors identifi ed WISP-1 (capable of inducing cartilage-degrading enzymes such as MMPs, ADAMTS-4 and ADAMTS-5), independently of the catabolic cytokine IL-1, as a crucial Wnt signaling mediator [62].
Moreover recent studies using a transgenic OA mouse model with conditional activation of the β-catenin gene in articular chondrocytes showed that upregulation of βcatenin signaling is most probably responsible for the conversion of normal articular chondrocytes into arthritic, OA-like cells. Chondrocyte maturational genes were activated along with the induction of matrix degradation [63,64]. Hedgehog signaling was also described to play a role in OA. Lin and colleagues demonstrated an increased expression of hedgehog targets in human OA samples and mouse articular cartilage after surgical OA induction. Amplifi ed hedge hog target gene Knee cartilage with higher Mankin scores shows increased hypertrophy (increase in collagen X staining; fast blue) and mineralization (arrowheads). Histologic assessment of the knee was performed on cartilage plugs from the medial femoral condyle, and a modifi ed Mankin scoring system was used to assess the severity of changes in osteoarthritis (OA) articular cartilage. The Mankin grades (range 0 to 14 points) for mild (1), moderate (2), and severe (3) OA were 2 to 5, 6 to 9, and 10 to 14 points, respectively. To assess hypertrophic diff erentiation, collagen X staining was performed on paraffi n sections after hyaluronidase (Sigma, Taufkirchen, Germany) digestion with collagen X antibody (ab58632; Abcam, Cambridge, UK) using the Vectastain ABC Elite Kit (Vector Laboratories, Peterborough, UK) and fast blue as a chromogen. To assess mineralization, the articular cartilage was analyzed using digital contact radiography (DCR), performed using a digital mammography imaging technique (Hologic, Waltham, MA, USA) operating at 25 kV in manual mode, usually at 3.8 mA, and with a fi lm focus distance of 8 cm. See [54]. Photographs kindly provided by Martin Fuerst, University Medical Center Hamburg-Eppendorf. expression correlated with advanced disease stages, and hedgehogs were described to stimulate the expression of the aggrecanase ADAMTS-5 via the transcription factor Runx2 [65].
Last, but not least, the retinoids display multiple eff ects relevant to the OA disease process. Davies and colleagues showed that components of the retinoid signaling pathways are upregulated during OA in humans and that all-trans-retinoic acid treatment of human explant cartilage samples led to signifi cant increase of MMP-13 and aggrecanases, enzymes involved in two of the key proteolytic processes implicated in OA [66]. Taken together, many locally acting growth factors playing a crucial role in the regulation of chondrocyte proliferation and diff erentiation during EO also are involved in OA pathogenesis and disease progression, mainly by stimu lation or activation of degradative enzymes (for example, MMP-13 or aggrecanases).

Role of hormones in osteoarthritis
A number of diff erent studies have attempted to link changes in hormonal status to the pathogenesis or progression of OA, but in the majority of cases inconsistent results were obtained. Th e prevalence of knee OA, for example, is increased among woman after the age of 50 years, and this phenomenon has been ascribed to estrogen insuffi ciency. No clear association has yet been found, however, between hormone defi ciency and OA of the hand, hip and knee [67]. Reports about estrogen replacement therapies and their outcome on OA incidence also show inconsistent results [68]. In addition to estrogen, the vitamin D status is unrelated to the risk of joint space or cartilage loss in knee OA [69]. Moreover, no association was found between vitamin D receptor polymorphisms and OA susceptibility in a large meta-analysis [70].
With respect to the GH/IGF-I axis, however, it was shown in a rat model of OA that chronic GH defi ciency causes an increased severity of articular cartilage lesions of OA, although the IGF-I expression is increased [71]. Anabolic IGF-I signaling is antagonized by increased occurrence of IGF-binding proteins, which then negatively regulate IGF-I signaling in chondrocytes during OA [72]. In contrast, patients with GH defi ciency had signifi cantly less OA than the normal patients of a control population, suggesting GH to be a benefi cial factor in the development of OA [73].

Role of extracellular matrix molecules in osteoarthritis
Cell-matrix interactions are essential regulators, both in EO and OA. One such example is defi ciency in the collagen IX α 1 chain, a perifi brillar component of cartilage fi brils, containing collagens II and XI. Knockout animals were not only shown to have a growth plate phenotype but also to develop a severe degenerative joint disease resembling human OA [74,75]. Histological analysis reveals OA-like changes in an age-dependent manner in the knee and temporomandibular joints starting at the age of 3 months. Later, at the age of 6 months, enhanced proteoglycan and collagen degradation due to higher expression of MMP-13 was observed. Th e FACIT collagen IX is directly and indirectly involved in the mutual interaction of the extrafi brillar matrix components with cartilage fi brils [76]. Th e disturbed tissue integrity possibly triggers a higher susceptibility of collagen IX α 1 knockout animals to cartilage degradation.
Furthermore, as in EO, matrix receptors binding to the structural components of the ECM have an impact on chondrocyte behavior in articular cartilage. One such example is the defi ciency of the α 1 subunit of integrins, which in mice was detected to be associated with an accelerated, aging-dependent development of OA [77]. Mice with a conditional deletion of the β 1 -integrin gene in early limb development using a mitochondrial peroxiredoxin Prx1-cre transgene showed multiple abnormalities of knee-joint articular cartilage, accompanied by accelerated terminal diff erentiation. Th e cartilage homeo stasis in these mice, however, was comparable with wild-type animals, suggesting a minor importance of signaling events mediated through integrins in cartilage destruction [78].
Other examples aff ecting chondrocyte behavior, however, are the DDR receptors. Xu and colleagues detected that increased expression of the collagen receptor DDR-2 in articular cartilage represents a key event in the pathogenesis of OA [79]. Th e authors not only describe increased immunostaining for DDR-2 but also for MMP-13 and MMP-derived type II collagen fragments in cartilage from patients with OA and from mice with surgically induced OA, and they linked the enhanced MMP-13 expression by mutation analysis directly to enhanced DDR-2 signaling. Based on these observations, they hypothesized that exposure of the type II collagen network to chondrocytes results in enhanced contact of the cells with type II collagen fi brils. DDR-2 is activated as a consequence of the interaction of type II collagen with chondrocytes, resulting in the increased expression of the receptor itself as well as MMP-13. Increased expression of DDR-2 may thus be a common event in the pathogenesis of OA in general [79].

Role of proteases in osteoarthritis
Proteolytic degradation of articular cartilage involving the key players of the MMP family, the a disintegrin and metalloprotease (ADAM) family and the ADAMTS protease family is a central event during OA. Th e structural integrity of the ECM is damaged by these enzyme activities, and cell-matrix interactions infl uencing chondrocyte activities are destroyed.
Although ADAMTS-4 expression is upregulated in human OA, only the lack of ADAMTS-5 prevented cartilage degradation in a mouse model of surgically induced OA [80]. Remarkably, the heparan sulfate proteo glycan syndecan-4 regulates ADAMTS-5 activation and cartilage breakdown in murine OA through direct interaction with the protease and through regulating mitogen-activated protein kinase-dependent synthesis of MMP-3 [81]. To investigate the role of MMP-13 in cartilage degradation and chondrocyte diff er entiation during OA, Little and colleagues surgically induced OA in the knees of MMP-13-defi cient and wildtype mice. Th ese authors observed that MMP-13 defi ciency can inhibit cartilage erosion but not chondrocyte hypertrophy or osteophyte generation during OA, suggesting that chondrocyte hypertrophy is accompanied by, but not directly regulated by, MMP-13 [82]. Tchetina and colleagues investigated the interrelationship between the extent of collagen cleavage by collagenases and the expression of diff erentiation-related genes [83]. Th ese  authors detected that early focal cartilage degradation by MMP-1, MMP-14 and ADAMTS-5 was accompanied by the expression of terminal diff erentiation-related genes COL10A1, MMP-13, MMP-9, Ihh and caspase 3, suggesting that chondrocyte diff erentiation may be closely related to the very early development of cartilage degeneration. Taken together these results indicate that matrix remodeling processes show similar characteristics in EO and OA.

Role of transcription factors in osteoarthritis
Orfanidou and colleagues recently investigated the involvement of the most fundamental transcription factors in EO -Runx2 and Sox9 -in the regulation of osteoarthritic chondrocytes. Th e authors demonstrated convincing associations among Runx2, Sox9 and FGF-23 in relation to MMP-13 expression in osteoarthritic chondrocytes, contributing to the cartilage degeneration process [84]. Kamekura and colleagues surgically induced OA in Runx2 +/--defi cient mice and wild-type mice. Th e heterozygous Runx2-defi cient mice exhibited decreased cartilage destruction and osteophyte formation, along with reduced type X collagen and MMP-13 expression, as compared with wild-type mice -suggesting a contribution of the transcription factor Runx2 to the pathogenesis of OA through chondrocyte hypertrophy and matrix breakdown after the induction of joint instability [85]. Taken together, these two major transcriptional regulators of chondrogenesis and hypertrophic diff erentiation (Sox9 and Runx2) play a role not only in bone development by EO but also in cartilage degradation in OA.
Th e transcription factor C/EBPβ was shown to directly transactivate p57 Kip2 to promote the transition from proliferation to hypertrophic diff erentiation of chondrocytes and to infl uence the collagen type X expression during bone development [46,48]. Th is transcription factor mediates cartilage destruction during OA progression, since C/EBPβ +/mice were protected against cartilage degradation in knee joints in an OA model [46]. Whether GADD45β is an upstream modulator of C/EBPβ in this instability-induced OA in mice, as was shown in chondrocyte terminal diff erentiation, needs to be shown in future experiments.

Conclusions
A number of signaling factors involved in chondrocyte proliferation and diff erentiation during EO were also shown to play a regulative role in articular cartilage during OA, pointing towards analogous signaling events that are critical for both scenarios (Table 1). All events leading to a structurally altered ECM in articular cartilage -for example, reduction in cartilage collagen production or induction of degradative enzymes -have to be taken into account as the driving force in the pathogenesis or progression of OA. Future work is necessary to investigate both of these processes in further detail in order to take advantage of the understanding of developmental aspects for pathogenetic mechanisms of degenerative joint disorders, and hence the successful development of future therapeutic strategies.