Biological impact of the fibroblast growth factor family on articular cartilage and intervertebral disc homeostasis

Two members of the fibroblast growth factor (FGF) family, basic FGF (bFGF) and FGF-18, have been implicated in the regulation of articular and intervertebral disc (IVD) cartilage homeostasis. Studies on bFGF from a variety of species have yielded contradictory results with regards to its precise role in cartilage matrix synthesis and degradation. In contrast, FGF-18 is a well-known anabolic growth factor involved in chondrogenesis and articular cartilage repair. In this review, we examined the biological actions of bFGF and FGF-18 in articular and IVD cartilage, the specific cell surface receptors bound by each factor, and the unique signaling cascades and molecular pathways utilized to exert their biological effects. Evidence suggests that bFGF selectively activates FGF receptor 1 (FGFR1) to exert degradative effects in both human articular chondrocytes and IVD tissue via upregulation of matrix-degrading enzyme activity, inhibition of matrix production, and increased cell proliferation resulting in clustering of cells seen in arthritic states. FGF-18, on the other hand, most likely exerts anabolic effects in human articular chondrocytes by activating FGFR3, increasing matrix formation and cell differentiation while inhibiting cell proliferation, leading to dispersed cells surrounded by abundant matrix. The results from in vitro and in vivo studies suggest the potential usefulness of bFGF and FGFR1 antagonists, as well as FGF-18 and FGFR3 agonists, as potential therapies to prevent cartilage degeneration and/or promote cartilage regeneration and repair in the future.     

Fibroblast growth factor control of cartilage homeostasis

Osteoarthritis (OA) and degenerative disc disease (DDD) are similar diseases involving the breakdown of cartilage tissue, and a better understanding of the underlying biochemical processes involved in cartilage degeneration may allow for the development of novel biologic therapies aimed at slowing the disease process. Three members of the fibroblast growth factor (FGF) family, FGF‐2, FGF‐18, and FGF‐8, have been implicated as contributing factors in cartilage homeostasis. The role of FGF‐2 is controversial in both articular and intervertebral disc (IVD) cartilage as it has been associated with species‐ and age‐dependent anabolic or catabolic events. Recent evidence suggests that FGF‐2 selectively activates FGF receptor 1 (FGFR1) to exert catabolic effects in human articular chondrocytes and IVD tissue via upregulation of matrix‐degrading enzyme production, inhibition of extracellular matrix (ECM) accumulation and proteoglycan synthesis, and clustering of cells characteristic of arthritic states. FGF‐18, on the other hand, most likely exerts anabolic effects in human articular chondrocytes by activating the FGFR3 pathway, inducing ECM formation and chondrogenic cell differentiation, and inhibiting cell proliferation. These changes result in dispersed chondrocytes or disc cells surrounded by abundant matrix. The role of FGF‐8 has recently been identified as a catabolic mediator in rat and rabbit articular cartilage, but its precise biological impact on human adult articular cartilage or IVD tissue remains unknown. The available evidence reveals the promise of FGF‐2/FGFR1 antagonists, FGF‐18/FGFR3 agonists, and FGF‐8 antagonists (i.e., anti‐FGF‐8 antibody) as potential therapies to prevent cartilage degeneration and/or promote cartilage regeneration and repair in the future. J. Cell. Biochem. 114: 735–742, 2013. © 2012 Wiley Periodicals, Inc.     

FGF18 is required for early chondrocyte proliferation, hypertrophy and vascular invasion of the growth plate

Fibroblast growth factor 18 (FGF18) has been shown to regulate chondrocyte proliferation and differentiation by signaling through FGF receptor 3 (FGFR3) and to regulate osteogenesis by signaling through other FGFRs. Fgf18(-/-) mice have an apparent delay in skeletal mineralization that is not seen in Fgfr3(-/-) mice. However, this delay in mineralization could not be simply explained by FGF18 signaling to osteoblasts. Here we show that delayed mineralization in Fgf18(-/-) mice was closely associated with delayed initiation of chondrocyte hypertrophy, decreased proliferation at early stages of chondrogenesis, delayed skeletal vascularization and delayed osteoclast and osteoblast recruitment to the growth plate. We further show that FGF18 is necessary for Vegf expression in hypertrophic chondrocytes and the perichondrium and is sufficient to induce Vegf expression in skeletal explants. These findings support a model in which FGF18 regulates skeletal vascularization and subsequent recruitment of osteoblasts/osteoclasts through regulation of early stages of chondrogenesis and VEGF expression. FGF18 thus coordinates neovascularization of the growth plate with chondrocyte and osteoblast growth and differentiation.     

Fibroblast growth factor 21 (FGF21) inhibits chondrocyte function and growth hormone action directly at the growth plate

Fibroblast growth factor 21 (FGF21) modulates glucose and lipid metabolism during fasting. In addition, previous evidence indicates that increased expression of FGF21 during chronic food restriction is associated with reduced bone growth and growth hormone (GH) insensitivity. In light of the inhibitory effects on growth plate chondrogenesis mediated by other FGFs, we hypothesized that FGF21 causes growth inhibition by acting directly at the long bones' growth plate. We first demonstrated the expression of FGF21, FGFR1 and FGFR3 (two receptors known to be activated by FGF21) and β-klotho (a co-receptor required for the FGF21-mediated receptor binding and activation) in fetal and 3-week-old mouse growth plate chondrocytes. We then cultured mouse growth plate chondrocytes in the presence of graded concentrations of rhFGF21 (0.01-10 μg/ml). Higher concentrations of FGF21 (5 and 10 μg/ml) inhibited chondrocyte thymidine incorporation and collagen X mRNA expression. 10 ng/ml GH stimulated chondrocyte thymidine incorporation and collagen X mRNA expression, with both effects prevented by the addition in the culture medium of FGF21 in a concentration-dependent manner. In addition, FGF21 reduced GH binding in cultured chondrocytes. In cells transfected with FGFR1 siRNA or ERK 1 siRNA, the antagonistic effects of FGF21 on GH action were all prevented, supporting a specific effect of this growth factor in chondrocytes. Our findings suggest that increased expression of FGF21 during food restriction causes growth attenuation by antagonizing the GH stimulatory effects on chondrogenesis directly at the growth plate. In addition, high concentrations of FGF21 may directly suppress growth plate chondrocyte proliferation and differentiation.     

Signaling mechanisms leading to the regulation of differentiation and apoptosis of articular chondrocytes by insulin-like growth factor-1

Cartilage development is initiated by the differentiation of mesenchymal cells into chondrocytes. Differentiated chondrocytes in articular cartilage undergo dedifferentiation and apoptosis during arthritis, in which NO production plays a critical role. Here, we investigated the roles and mechanisms of action of insulin-like growth factor-1 (IGF-1) in the chondrogenesis of mesenchymal cells and the maintenance and survival of differentiated articular chondrocytes. IGF-1 induced chondrogenesis of limb bud mesenchymal cells during micromass culture through the activation of phosphatidylinositol 3-kinase (PI3K) and Akt. PI3K activation is required for the activation of protein kinase C (PKC)-alpha and p38 kinase and inhibition of ERK1/2. These events are necessary for chondrogenesis. The growth factor additionally blocked NO-induced dedifferentiation and apoptosis of primary culture articular chondrocytes. NO production in chondrocytes induced down-regulation of PI3K and Akt activities, which was blocked by IGF-1 treatment. Stimulation of PI3K by IGF-1 resulted in blockage of NO-induced activation of p38 kinase and ERK1/2 and inhibition of PKCalpha and PKCzeta, which in turn suppressed dedifferentiation and apoptosis. Our results collectively indicate that IGF-1 regulates differentiation, maintenance of the differentiated phenotype, and apoptosis of articular chondrocytes via a PI3K pathway that modulates ERK, p38 kinase, and PKC signaling.     

FGF upregulates osteopontin in epiphyseal growth plate chondrocytes: Implications for endochondral ossification

Fibroblast growth factor receptor 3 (FGFR3) signaling pathways are essential for normal longitudinal bone growth. Mutations in this receptor lead to various human growth disorders, including Achondroplasia, disproportionately short-limbed dwarfism, characterized by narrowing of the hypertrophic region of the epiphyseal growth plates. Here we find that FGF9, a preferred ligand for FGFR3 rapidly induces the upregulation and secretion of the matrix resident phosphoprotein, osteopontin (OPN) in cultured chicken chondrocytes. This effect was observed as early as two hours post stimulation and at FGF9 concentrations as low as 1.25 ng/ml at both mRNA and protein levels. OPN expression is known to be associated with chondrocyte and osteoblast differentiation and osteoclast activation. Unexpectedly, FGF9 induced OPN was accompanied by inhibition of differentiation and increased proliferation of the treated chondrocytes. Moreover, FGF9 stimulated OPN expression irrespective of the differentiation stage of the cells or culture conditions. In situ hybridization analysis of epiphyseal growth plates from chicken or mice homozygous for the Achondroplasia, G369C/mFGFR3 mutation demonstrated co-localization of OPN expression and osteoclast activity, as evidenced by tartarate resistant acid phosphatase positive cells in the osteochondral junction. We propose that FGF signaling directly activates OPN expression independent of chondrocytes differentiation. This may enhance the recruitment and activation of osteoclasts, and increase in cartilage resorption and remodeling in the chondro-osseus border.     

Fibroblast growth factor 2: good or bad guy in the joint?

Fibroblast growth factor 2 (FGF2) is a highly abundant growth factor found within the pericellular matrix of articular chondrocytes, but studies investigating its role have been conflicting. The paper reported by Yan and colleagues in the previous issue of Arthritis Research & Therapy proposes that differences in responses to FGF2 are most likely due to changes in the balance between the two major articular cartilage FGF receptors, FGFR1 and FGFR3. They show that the catabolic and anti-anabolic effects of FGF2 are mediated primarily through FGFR1 whereas the beneficial effects are through FGFR3. This balance is dynamic and is altered in disease and following growth factor stimulation in vitro.     

Ectodermal FGFs induce perinodular inhibition of limb chondrogenesis in vitro and in vivo via FGF receptor 2

The formation of cartilage elements in the developing vertebrate limb, where they serve as primordia for the appendicular skeleton, is preceded by the appearance of discrete cellular condensations. Control of the size and spacing of these condensations is a key aspect of skeletal pattern formation. Limb bud cell cultures grown in the absence of ectoderm formed continuous sheet-like masses of cartilage. With the inclusion of ectoderm, these cultures produced one or more cartilage nodules surrounded by zones of noncartilaginous mesenchyme. Ectodermal fibroblast growth factors (FGF2 and FGF8), but not a mesodermal FGF (FGF7), substituted for ectoderm in inhibiting chondrogenic gene expression, with some combinations of the two ectodermal factors leading to well-spaced cartilage nodules of relatively uniform size. Treatment of cultures with SU5402, an inhibitor FGF receptor tyrosine kinase activity, rendered FGFs ineffective in inducing perinodular inhibition. Inhibition of production of FGF receptor 2 (FGFR2) by transfection of wing and leg cell cultures with antisense oligodeoxynucleotides blocked appearance of ectoderm- or FGF-induced zones of perinodular inhibition of chondrogenesis and, when introduced into the limb buds of developing embryos, led to shorter, thicker, and fused cartilage elements. Because FGFR2 is expressed mainly at sites of precartilage condensation during limb development in vivo and in vitro, these results suggest that activation of FGFR2 by FGFs during development elicits a lateral inhibitor of chondrogenesis that limits the expansion of developing skeletal elements.     

Co-ordination of TGF-beta and FGF signaling pathways in bone organ cultures

Transforming growth factor-beta (TGF-beta) is known to regulate chondrocyte proliferation and hypertrophic differentiation in embryonic bone cultures by a perichondrium dependent mechanism. To begin to determine which factors in the perichondrium mediate the effects of TGF-beta, we studied the effect of Insulin-like Growth Factor-1 (IGF-I) and Fibroblast Growth Factors-2 and -18 (FGF2, FGF18) on metatarsal organ cultures. An increase in chondrocyte proliferation and hypertrophic differentiation was observed after treatment with IGF-I. A similar effect was seen after the perichondrium was stripped from the metatarsals suggesting IGF-I acts directly on the chondrocytes. Treatment with FGF-2 or FGF-18 resulted in a decrease in bone elongation as well as hypertrophic differentiation. Treatment also resulted in a decrease in BrdU incorporation into chondrocytes and an increase in BrdU incorporation in perichondrial cells, similar to what is seen after treatment with TGF-beta1. A similar effect was seen with FGF2 after the perichondrium was stripped suggesting that, unlike TGF-beta, FGF2 acts directly on chondrocytes to regulate proliferation and hypertrophic differentiation. To test the hypothesis that TGF-beta regulates IGF or FGF signaling, activation of the receptors was characterized after treatment with TGF-beta. Activation was measured as the level of tyrosine phosphorylation on the receptor. Treatment with TGF-beta for 24h did not alter the level of IGFR-I tyrosine phosphorylation. In contrast, treatment with TGF-beta resulted in and increase in tyrosine phosphorylation on FGFR3 without alterations in total FGFR3 levels. TGF-beta also stimulated expression of FGF18 mRNA in the cultures and the effects of TGF-beta on metatarsal development were blocked or partially blocked by pretreatment with FGF signaling inhibitors. The results suggest a model in which FGF through FGFR3 mediates some of the effects of TGF-beta on embryonic bone formation.     

BMPs regulate multiple aspects of growth-plate chondrogenesis through opposing actions on FGF pathways

Bone morphogenetic protein (BMP) signaling pathways are essential regulators of chondrogenesis. However, the roles of these pathways in vivo are not well understood. Limb-culture studies have provided a number of essential insights, including the demonstration that BMP pathways are required for chondrocyte proliferation and differentiation. However, limb-culture studies have yielded contradictory results; some studies indicate that BMPs exert stimulatory effects on differentiation, whereas others support inhibitory effects. Therefore, we characterized the skeletal phenotypes of mice lacking Bmpr1a in chondrocytes (Bmpr1a(CKO)) and Bmpr1a(CKO);Bmpr1b+/- (Bmpr1a(CKO);1b+/-) in order to test the roles of BMP pathways in the growth plate in vivo. These mice reveal requirements for BMP signaling in multiple aspects of chondrogenesis. They also demonstrate that the balance between signaling outputs from BMP and fibroblast growth factor (FGF) pathways plays a crucial role in the growth plate. These studies indicate that BMP signaling is required to promote Ihh expression, and to inhibit activation of STAT and ERK1/2 MAPK, key effectors of FGF signaling. BMP pathways inhibit FGF signaling, at least in part, by inhibiting the expression of FGFR1. These results provide a genetic in vivo demonstration that the progression of chondrocytes through the growth plate is controlled by antagonistic BMP and FGF signaling pathways.     

A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation

Activating mutations in FGF receptor 3 (FGFR3) cause several human dwarfism syndromes by affecting both chondrocyte proliferation and differentiation. Using microarray and biochemical analyses of FGF-treated rat chondrosarcoma chondrocytes, we show that FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the rapid dephosphorylation of the retinoblastoma protein (pRb) p107 and repression of a subset of E2F target genes by a mechanism that is independent of cyclin E-Cdk inhibition. In contrast, hypophosphorylation of pRb and p130 occur after growth arrest is first detected, and may contribute to its maintenance. Importantly, we also find a number of gene expression changes indicating that FGF promotes many aspects of hypertrophic differentiation, a notion supported by in situ analysis of developing growth plates from mice expressing an activated form of FGFR3. Thus, FGF may coordinate the onset of differentiation with chondrocyte growth arrest in the developing growth plate.     

Distribution of cartilage molecules in the developing mouse joint.

This study describes the precise spatial and temporal patterns of protein distribution for aggrecan, fibromodulin, cartilage oligomeric matrix protein (COMP) and cartilage matrix protein (CMP) in the developing mouse limb with particular attention to those cells destined to form articular chondrocytes in comparison to those cells destined to form a mineralized tissue and become replaced by bone. Mouse glenohumeral joints from fetal mice (12-18 days post coitus (dpc) to the young adult (37 days after birth) were immunostained with antibodies specific for these molecules. Aggrecan staining defined the general chondrocytic phenotype, whether articular or transient. Fibromodulin was associated with prechondrocytic mesenchymal cells in the interzone prior to joint cavitation and with the mesenchymal cells of the perichondrium or the periosteum encapsulating the joint elements of the maturing and young adult limb. Staining was most intense around developing articular chondrocytes and much less abundant or absent in those differentiating cells along the anlage. CMP showed an almost reciprocal staining pattern to fibromodulin and was not detected in the matrix surrounding articular chondrocytes. COMP was not detected in the cells at the articular surface prior to cavitation but by 18 dpc, as coordinated movement of the mouse forelimb intensifies, staining for COMP was most intense around the maturing articular chondrocytes. These results show that the cells that differentiate into articular chondrocytes elaborate an extracellular matrix distinct from those cells that are destined to form bone. Fibromodulin may function in the early genesis of articular cartilage and COMP may be associated with elaboration of a weight-bearing chondrocyte matrix.     

Growth factor transgenes interactively regulate articular chondrocytes

Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin‐like growth factor I (IGF‐I), fibroblast growth factor‐2 (FGF‐2), transforming growth factor beta1 (TGF‐β1), bone morphogenetic protein‐2 (BMP‐2), and bone morphogenetic protien‐7 (BMP‐7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF‐I and FGF‐2 maximized cell proliferation. The three‐transgene group encoding IGF‐I, BMP‐2, and BMP‐7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell‐based articular cartilage repair. J. Cell. Biochem. 114: 908–919, 2013. © 2012 Wiley Periodicals, Inc.     

Peptide growth factors and their interactions during chondrogenesis

Peptide growth factors have been implicated in three aspects of cartilage growth and metabolism; the induction of mesoderm and differentiation of a cartilaginous skeleton in the early embryo, the growth and differentiation of chondrocytes within the epiphyseal growth plates leading to endochondral calcification, and the processes of articular cartilage damage and repair. Three peptide growth factor classes have been strongly implicated in these processes, the fibroblast growth factor family (FGF), the insulin-like growth factors (IGFs) including insulin, and transforming growth factor-β (TGF-β) and related molecules. Each of these peptide groups are expressed in the early embryo. Basic FGF, TGF-β and the related activin have been shown to induce the appearance of mesoderm from primitive neuroectoderm. TGF-β and related bone morphometric proteins can induce the differentiation of cartilage from primitive mesenchyme, and together with basic FGF and IGFs promote cartilage growth. Each class of growth factor is expressed within the epiphyseal growth plate where their autocrine/paracrine interactions regulate the rate of chondrocyte proliferation, matrix protein synthesis and terminal differentiation and mineralization. Basic FGF may prove useful in articular cartilage repair, while basic FGF, IGFs and TGF-β are among a number of growth factors and cytokines that have been implicated in cartilage disease.     

Sulfoxide stimulation of chondrogenesis in limb mesenchyme is accompanied by an increase in type II collagen enhancer activity

We have utilized a modification of the limb bud mesenchyme micromass culture system to screen compounds that might stimulate chondrogenesis. Two compounds in the sulfoxide family (methylphenylsulfoxide and p-chlorophenyl methyl sulfoxide) were stimulatory at 10(-2) M and 10(-3) M, respectively; whereas other sulfoxides and organic solvents were not active at these concentrations. In addition, specific growth factors (basic FGF, IGF-I, IGF-II) were not chondroinductive at concentrations that are active in other cell systems. Both sulfoxide compounds stimulated cartilage nodule formation, [35S]sulfate incorporation, and activity of the regulatory sequences of the collagen II gene. In contrast, transforming growth factor beta-1 (10 ng/ml) stimulated sulfate incorporation but produced only a diffuse deposition of cartilage matrix and reduced the ability of the cells to utilize the regulatory sequences of the collagen II gene. The sulfoxides appear to promote the differentiation of limb bud cells to chondrocytes and thus exhibit chondroinductive activity.     

Regulation of osteoblast, chondrocyte, and osteoclast functions by fibroblast growth factor (FGF)-18 in comparison with FGF-2 and FGF-10.

This study investigated the actions of fibroblast growth factor (FGF)-18, a novel member of the FGF family, on osteoblasts, chondrocytes, and osteoclasts and compared them with those of FGF-2 and FGF-10. FGF-18 stimulated the proliferation of cultured mouse primary osteoblasts, osteoblastic MC3T3-E1 cells, primary chondrocytes, and prechondrocytic ATDC5 cells, although it inhibited the differentiation and matrix synthesis of these cells. FGF-18 up-regulated the phosphorylation of extracellular signal-regulated kinase in both osteoblasts and chondrocytes and up-regulated the phosphorylation of p38 mitogen-activated protein kinase only in chondrocytes. FGF-18 mitogenic actions were blocked by a specific inhibitor of extracellular signal-regulated kinase in both osteoblasts and chondrocytes and by a specific inhibitor of p38 mitogen-activated protein kinase in chondrocytes. With regard to the action of FGF-18 on bone resorption, FGF-18 not only induced osteoclast formation through receptor activator of nuclear factor-kappaB ligand and cyclooxygenase-2 but also stimulated osteoclast function to form resorbed pits on a dentine slice in the mouse coculture system. All these effects of FGF-18 bore a close resemblance to those of FGF-2, whereas FGF-10 affects none of these cells. FGF-18 may therefore compensate for the action of FGF-2 on bone and cartilage.