Fibroblast growth factor (FGF) 18 signals through FGF receptor 3 to promote chondrogenesis

Signaling by fibroblast growth factor (FGF) 18 and FGF receptor 3 (FGFR3) have been shown to regulate proliferation, differentiation, and matrix production of articular and growth plate chondrocytes in vivo and in vitro. Notably, the congenital absence of either FGF18 or FGFR3 resulted in similar expansion of the growth plates of fetal mice and the addition of FGF18 to human articular chondrocytes in culture enhanced proliferation and matrix production. Based on these and other experiments it has been proposed that FGF18 signals through FGFR3 to promote cartilage production by chondrocytes. Its role in chondrogenesis remains to be defined. In the current work we used the limb buds of FGFR3(+/+) and FGFR3(-/-) embryonic mice as a source of mesenchymal cells to determine how FGF18 signaling affects chondrogenesis. Confocal laser-scanning microscopy demonstrated impaired cartilage nodule formation in the FGFR3(-/-) cultures. Potential contributing factors to the phenotype were identified as impaired mitogenic response to FGF18, decreased production of type II collagen and proteoglycan in response to FGF18 stimulation, impaired interactions with the extracellular matrix resulting from altered integrin receptor expression, and altered expression of FGFR1 and FGFR2. The data identified FGF18 as a selective ligand for FGFR3 in limb bud mesenchymal cells, which suppressed proliferation and promoted their differentiation and production of cartilage matrix. This work, thus, identifies FGF18 and FGFR3 as potential molecular targets for intervention in tissue engineering aimed at cartilage repair and regeneration of damaged cartilage.     

Effects of diadenosine tetraphosphate on FGF9-induced chloride flux changes in achondroplastic chondrocytes

Achondroplasia, the most common type of dwarfism, is characterized by a mutation in the fibroblast growth factor receptor 3 (FGFR3). Achondroplasia is an orphan pathology with no pharmacological treatment so far. However, the possibility of using the dinucleotide diadenosine tetraphosphate (Ap₄A) with therapeutic purposes in achondroplasia has been previously suggested. The pathogenesis involves the constitutive activation of FGFR3, resulting in altered biochemical and physiological processes in chondrocytes. Some of these altered processes can be influenced by changes in cell volume and ionic currents. In this study, the action of mutant FGFR3 on chondrocyte size and chloride flux in achondroplastic chondrocytes was investigated as well as the effect of the Ap₄A on these processes triggered by mutant FGFR3. Stimulation with the fibroblast growth factor 9 (FGF9), the preferred ligand for FGFR3, induced an enlarged achondroplastic chondrocyte size and an increase in the intracellular chloride concentration, suggesting the blockade of chloride efflux. Treatment with the Ap₄A reversed the morphological changes triggered by FGF9 and restored the chloride efflux. These data provide further evidence for the therapeutic potential of this dinucleotide in achondroplasia treatment.     

Sulfated glycosaminoglycans mediate the effects of FGF2 on the osteogenic potential of rat calvarial osteoprogenitor cells

Fibroblast growth factor-2 (FGF2) is a powerful promoter of bone growth. We demonstrate here that brief exposure to FGF2 enhances mineralized nodule formation in cultured rat osteoprogenitor cells due to an expansion of cells that subsequently mineralize. This mitogenic effect is mediated via sulfated glycosaminoglycans (GAGs), FGFR1, and the extracellular signal-regulated kinase (ERK) pathway. The GAGs involved in this stimulation are chondroitin sulfates (CS) rather than heparan sulfates (HS). However, continuous FGF2 treatment reduces alkaline phosphatase (ALP) activity, downregulates collagen Ialpha1 (ColIalpha1) and FGFR3 expression, upregulates the expression and secretion of osteopontin (OPN) and inhibits mineralization. The inhibitory effects of FGF2 on FGFR3 expression and ALP activity are also mediated by the ERK pathway, although the effects of FGF2 on ColIalpha1 and OPN expression are mediated by GAGs and PKC activity. Thus short-term activation of FGF2/FGFR1 promotes osteoprogenitor proliferation and subsequent differentiation, while long-term activation of FGF2 signaling disrupts mineralization by modulating osteogenic marker expression. This study thus establishes the central role of sulfated GAGs in the osteogenic progression of osteoprogenitors.     

Gene disruption of Spred-2 causes dwarfism

The impact of the fibroblast growth factor receptor 3 (FGFR3)-mediated signaling pathway on bone growth has been demonstrated by various genetic approaches. Overexpression of fibroblast growth factors (FGFs), several gain-of-function mutations in the FGFR3, and constitutive activation of mitogen-activated protein kinase (MAPK) kinase (MEK1) in chondrocytes have been shown to cause dwarfism in mice by activation of the MAPK signaling pathway. To investigate the previously reported inhibitory role of Spred in the FGFR3/MAPK pathway, we generated mice with a trapped Spred-2 gene. Here we show that lack of functional Spred-2 protein in mice caused a dwarf phenotype, similar to achondroplasia, the most common form of human dwarfism. Spred-2(-/-) mice showed reduced growth and body weight, they had a shorter tibia length, and showed narrower growth plates as compared with wild-type mice. We detected promoter activity and protein expression of Spred-2 in chondrocytes, suggesting an important function of Spred-2 in chondrocytes and bone development. Stimulation of chondrocytes with different FGF concentrations showed earlier and augmented ERK phosphorylation in Spred-2(-/-) chondrocytes in comparison to Spred-2(+/+) chondrocytes. Our observations suggest a model in which loss of Spred-2 inhibits bone growth by inhibiting chondrocyte differentiation through up-regulation of the MAPK signaling pathway.     

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.     

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.     

Meclozine Facilitates Proliferation and Differentiation of Chondrocytes by Attenuating Abnormally Activated FGFR3 Signaling in Achondroplasia

Achondroplasia (ACH) is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS) cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8) cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP) as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias.     

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.     

Downregulation of Akt activity contributes to the growth arrest induced by FGF in chondrocytes

Unregulated FGF signaling produced by activating FGFR3 mutations causes several forms of dwarfism-associated chondrodysplasias in humans and mice. FGF signaling inhibits chondrocyte proliferation by activating multiple signal transduction pathways that all contribute to chondrocyte growth arrest and induction of some aspects of differentiation. Previous studies had identified the Stat1 pathway, dephosphorylation of the Rb family proteins p107 and p130, induction of p21 expression and sustained activation of MAP kinases as playing a role in the FGF response of chondrocytes. We have examined the role of Akt (PKB) in the response of chondrocytes to FGF signaling. Differently from what is observed in many other cell types, FGF does not activate Akt in chondrocytes, and Akt phosphorylation is actually downregulated after FGF treatment. By expressing a constitutively activated, myristylated form of Akt (myr-Akt) in the RCS chondrosarcoma cell line, we show that Akt activation partially counteracts the inhibitory effect of FGF signaling. The response of myr-Akt expressing cells to FGF is identical to parental RCS in the first few hours after treatment, but then diverges as myr-Akt cells show decreased p130 phosphorylation, increased cyclin E/cdk2 activity and continue to proliferate at a slow rate. Constitutive Akt activation does not affect p21 expression but appears to influence directly cdk/cyclin activity. On the other hand, the induction of differentiation-related genes is unchanged in myr-Akt cells. These results identify Akt downregulation as an important aspect of the response of chondrocytes to FGF that, however, only affects chondrocyte proliferation and not the ability of FGF to induce differentiation genes.     

Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway

Achondroplasia is the most common genetic form of human dwarfism, for which there is presently no effective therapy. C-type natriuretic peptide (CNP) is a newly identified molecule that regulates endochondral bone growth through GC-B, a subtype of particulate guanylyl cyclase. Here we show that targeted overexpression of CNP in chondrocytes counteracts dwarfism in a mouse model of achondroplasia with activated fibroblast growth factor receptor 3 (FGFR-3) in the cartilage. CNP prevented the shortening of achondroplastic bones by correcting the decreased extracellular matrix synthesis in the growth plate through inhibition of the MAPK pathway of FGF signaling. CNP had no effect on the STAT-1 pathway of FGF signaling that mediates the decreased proliferation and the delayed differentiation of achondroplastic chondrocytes. These results demonstrate that activation of the CNP-GC-B system in endochondral bone formation constitutes a new therapeutic strategy for human achondroplasia.     

Overexpression of Spry1 in chondrocytes causes attenuated FGFR ubiquitination and sustained ERK activation resulting in chondrodysplasia

The FGF signaling pathway plays essential roles in endochondral ossification by regulating osteoblast proliferation and differentiation, chondrocyte proliferation, hypertrophy, and apoptosis. FGF signaling is controlled by the complementary action of both positive and negative regulators of the signal transduction pathway. The Spry proteins are crucial regulators of receptor tyrosine kinase-mediated MAPK signaling activity. Sprys are expressed in close proximity to FGF signaling centers and regulate FGFR-ERK-mediated organogenesis. During endochondral ossification, Spry genes are expressed in prehypertrophic and hypertrophic chondrocytes. Using a conditional transgenic approach in chondrocytes in vivo, the forced expression of Spry1 resulted in neonatal lethality with accompanying skeletal abnormalities resembling thanatophoric dysplasia II, including increased apoptosis and decreased chondrocyte proliferation in the presumptive reserve and proliferating zones. In vitro chondrocyte cultures recapitulated the inhibitory effect of Spry1 on chondrocyte proliferation. In addition, overexpression of Spry1 resulted in sustained ERK activation and increased expression of p21 and STAT1. Immunoprecipitation experiments revealed that Spry1 expression in chondrocyte cultures resulted in decreased FGFR2 ubiquitination and increased FGFR2 stability. These results suggest that constitutive expression of Spry1 in chondrocytes results in attenuated FGFR2 degradation, sustained ERK activation, and up-regulation of p21Cip and STAT1 causing dysregulated chondrocyte proliferation and terminal differentiation.     

betaKlotho is required for fibroblast growth factor (FGF) 21 signaling through FGF receptor (FGFR) 1c and FGFR3c

Fibroblast growth factor (FGF) 21, a structural relative of FGF23 that regulates phosphate homeostasis, is a regulator of insulin-independent glucose transport in adipocytes and plays a role in the regulation of body weight. It also regulates ketogenesis and adaptive responses to starvation. We report that in a reconstituted receptor activation assay system using BaF3 cells, which do not endogenously express any type of FGF receptor (FGFR) or heparan sulfate proteoglycan, FGF21 alone does not activate FGFRs and that betaKlotho is required for FGF21 to activate two specific FGFR subtypes: FGFR1c and FGFR3c. Coexpression of betaKlotho and FGFR1c on BaF3 cells enabled FGF21, but not FGF23, to activate receptor signaling. Conversely, coexpression of FGFR1c and Klotho, a protein related to betaKlotho, enabled FGF23 but not FGF21 to activate receptor signaling, indicating that expression of betaKlotho/Klotho confers target cell specificity on FGF21/FGF23. In all of these cases, heparin enhanced the activation but was not essential. In 3T3-L1 adipocytes, up-regulation of glucose transporter (GLUT) expression by FGF21 was associated with expression of betaKlotho, which was absent in undifferentiated 3T3-L1 fibroblasts. It is thus suggested that betaKlotho expression is a crucial determinant of the FGF21 specificity of the target cells upon which it acts in an endocrine fashion.     

Expression of fibroblast growth factor receptor 3 by fibroblast growth factor 2 in cultured chick embryo chondrocytes

Although fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 3 (FGFR3) both inhibit longitudinal bone growth, little is known about the relationship between FGF2 and FGFR3. Accordingly, the current study examined the expression of FGFR3 mRNA after the administration of FGF2 using cultured chondrocytes from day 17 chick embryos to evaluate the relationship between FGF2 and FGFR3. The chondrocytes were isolated from the caudal one-third portion (LS) of sterna, peripheral regions (USP) and central core regions (USC) of the cephalic portion of the sterna, and lower portion of the proximal tibial growth plate (Ti) of day 17 chick embryo. The expression of FGFR1, FGFR3, and type II and X collagen mRNA in the chondrocytes from the LS, USP, USC, and Ti was determined. FGFR1 was not expressed in the LS and USP chondrocytes, yet strongly expressed in the USC and Ti chondrocytes. With a treatment of FGF2, the expression of FGFR1 slightly increased in the USC chondrocytes and was not related with the concentration of FGF2 in the Ti chondrocytes. FGFR3 was expressed in all the chondrocyte types, yet strongly increased in the LS, USC, USP, and Ti in that order according to the concentration of FGF2. For the LS and USP chondrocytes, the expression of FGFR3 with FGF2 increased in a 4-day culture, yet decreased in a 6-day culture, whereas for the USC chondrocytes, the expression of FGFR3 mRNA with FGF2 increased in a 2-day culture, yet decreased in a 4-day culture, suggesting that the hypertrophic chondrocytes were more numerous and sensitive compared to the proliferative chondrocytes. For all the chondrocyte types, FGF2 appeared to be up-regulated to FGFR3, as the expression of FGFR3 mRNA increased with a higher concentration of FGF2 until a peak level. In conclusion, FGF2 was found to up-regulate to FGFR3 until the peak level of FGFR3 mRNA expression, while in hypertrophic chondrocytes, FGFR3 appeared to cause the differentiaton of chondrocytes, resulting in the inhibition of longitudinal bone growth after the peak level of FGFR3 mRNA expression.     

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.     

Aggrecan modulation of growth plate morphogenesis

Chick and mouse embryos with heritable deficiencies of aggrecan exhibit severe dwarfism and premature death, demonstrating the essential involvement of aggrecan in development. The aggrecan-deficient nanomelic (nm) chick mutant E12 fully formed growth plate (GP) is devoid of matrix and exhibits markedly altered cytoarchitecture, proliferative capacity, and degree of cell death. While differentiation of chondroblasts to pre-hypertrophic chondrocytes (IHH expression) is normal up to E6, the extended periosteum expression pattern of PTCH (a downstream effector of IHH) indicates altered propagation of IHH signaling, as well as accelerated down-regulation of FGFR3 expression, decreased BrdU incorporation and higher levels of ERK phosphorylation, all indicating early effects on FGF signaling. By E7 reduced IHH expression and premature expression of COL10A1 foreshadow the acceleration of hypertrophy observed at E12. By E8, exacerbated co-expression of IHH and COL10A1 lead to delayed separation and establishment of the two GPs in each element. By E9, increased numbers of cells express P-SMAD1/5/8, indicating altered BMP signaling. These results indicate that the IHH, FGF and BMP signaling pathways are altered from the very beginning of GP formation in the absence of aggrecan, thereby inducing premature hypertrophic chondrocyte maturation, leading to the nanomelic long bone growth disorder.     

Fibroblast growth factor inhibits chondrocytic growth through induction of p21 and subsequent inactivation of cyclin E-Cdk2

Fibroblast growth factor (FGF) and its receptor (FGFR) are thought to be negative regulators of chondrocytic growth, as exemplified by achondroplasia and related chondrodysplasias, which are caused by constitutively active mutations in FGFR3. To understand the growth-inhibitory mechanisms of FGF, we analyzed the effects of FGF2 on cell cycle-regulating molecules in chondrocytes. FGF2 dramatically inhibited proliferation of rat chondrosarcoma (RCS) cells and arrested their cell cycle at the G(1) phase. FGF2 increased p21 expression in RCS cells, which assembled with the cyclin E-Cdk2 complexes, although the expression of neither cyclin E nor Cdk2 increased. In addition, the kinase activity of immunoprecipitated cyclin E or Cdk2, assessed with retinoblastoma protein (pRb) as substrate, was dramatically reduced by FGF-2. Moreover, FGF2 shifted pRb to its underphosphorylated, active form in RCS cells. FGF2 not only induced p21 protein expression in proliferating chondrocytes in mouse fetal limbs cultured in vitro but also decreased their proliferation as assessed by the expression of histone H4 mRNA, a marker for cells in S phase. Furthermore, inhibitory effects of FGF2 on chondrocytic proliferation were partially reduced in p21-null limbs, compared with those in wild-type limbs in vitro. Taken together, FGF's growth inhibitory effects of chondrocytes appear to be mediated at least partially through p21 induction and the subsequent inactivation of cyclin E-Cdk2 and activation of pRb.