ReviewDevelopmental Biology

Fibroblast Growth Factor Receptor Signaling Crosstalk in Skeletogenesis

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Science Signaling  02 Nov 2010:
Vol. 3, Issue 146, pp. re9
DOI: 10.1126/scisignal.3146re9

Abstract

Fibroblast growth factors (FGFs) play important roles in the control of embryonic and postnatal skeletal development by activating signaling through FGF receptors (FGFRs). Germline gain-of-function mutations in FGFR constitutively activate FGFR signaling, causing chondrocyte and osteoblast dysfunctions that result in skeletal dysplasias. Crosstalk between the FGFR pathway and other signaling cascades controls skeletal precursor cell differentiation. Genetic analyses revealed that the interplay of WNT and FGFR1 determines the fate and differentiation of mesenchymal stem cells during mouse craniofacial skeletogenesis. Additionally, interactions between FGFR signaling and other receptor tyrosine kinase networks, such as those mediated by the epidermal growth factor receptor and platelet-derived growth factor receptor α, were associated with excessive osteoblast differentiation and bone formation in the human skeletal dysplasia called craniosynostosis, which is a disorder of skull development. We review the roles of FGFR signaling and its crosstalk with other pathways in controlling skeletal cell fate and discuss how this crosstalk could be pharmacologically targeted to correct the abnormal cell phenotype in skeletal dysplasias caused by aberrant FGFR signaling.

Fibroblast Growth Factor Signaling

Fibroblast growth factor (FGF) signaling cascades are implicated in the control of several processes, including cell proliferation, differentiation, and apoptosis (1, 2). FGFs mediate their cellular responses by activating receptor tyrosine kinases (RTKs) called FGF receptors 1 to 4 (FGFR1–4). FGF binding to the extracellular domain of FGFR induces receptor dimerization and phosphorylation of tyrosine residues in the FGFR cytoplasmic domain. This leads to phosphorylation of signaling proteins and results in the activation of intracellular signaling pathways, such as phospholipase C (PLC), protein kinase C (PKC), Ras to mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) to Akt (PI3K/Akt) pathways (2, 3). The abundance of activated RTKs at the plasma membrane is regulated by negative feedback mechanisms involving receptor internalization and degradation that attenuate growth factor signaling (4). This down-regulation process involves several proteins, such as the ubiquitin ligase Cbl, an adaptor protein that is phosphorylated and recruited to various activated RTKs, the FGFRs, the epidermal growth factor receptors (EGFRs), and the platelet-derived growth factor receptors (PDGFRs) (5, 6). Cbl down-regulates RTKs by mediating their ubiquitination after ligand binding, resulting in proteasome-mediated RTK degradation. The docking protein FGFR substrate 2 (FRS2) is also involved in the down-regulation of the FGFR. In response to FGF stimulation, tyrosine-phosphorylated FRS2α forms a ternary complex with Cbl and Grb2, resulting in ubiquitination of FGFR and FRS2α (7, 8).

FGF-FGFR Signaling Pathways in Skeletogenesis

Skeletogenesis occurs both during embryonic development and postnatal life and is a complex process, involving the commitment of precursor mesenchymal cells that progressively differentiate into cartilage-forming cells (chondrocytes) or bone-forming cells (osteoblasts) (9). These differentiation paths are controlled by distinct transcription factors (10, 11). Both the number and activity of skeletal cells are regulated by multiple extracellular signaling molecules, including the morphogens bone morphogenetic proteins (BMPs) and Wnt (12, 13) and the growth factor FGF (14, 15). In mammals, during development FGFs, FGFR1–3, and their co-receptors (heparin sulfate proteoglycans) are present in the mesenchyme, chondrocytes, or osteoblasts (1618). Genetic and functional studies in mice showed that FGFs are important regulators of chondrocyte and osteoblast differentiation (19, 20) and that this involves the activation of FGFRs and multiple signaling mechanisms (21). In cartilage, FGFR signaling stimulates extracellular signal–regulated kinases 1 and 2 (ERK1/2), p38 MAPK, PLCγ-mediated activation of STAT1, and PI3K/Akt, as well as BMP and WNT signaling, to inhibit chondrocyte proliferation and increase differentiation and apoptosis. Signaling molecules activated by FGFR2 in osteoblasts include ERK1/2, PKC, Src (nonreceptor tyrosine kinase), and PI3K/Akt, resulting in increased expression of several genes involved in osteogenesis. Furthermore, some of these signaling pathways may converge to promote genes involved in osteogenesis (14, 21).

Promotion of Osteogenesis by Activating FGFR Mutations

The important role of FGF/FGFR signaling in skeletogenesis is supported by the observation that mutations in FGFR1–3 lead to severe impairment in skeletal development (22). Activating mutations in FGFR3 result in chondrodysplasias and dwarfism. Mutations in FGFR1 or 2 cause craniosynostosis, which results in skeletal dysplasias characterized by premature fusion of one or more cranial sutures, such as occur in Apert, Crouzon, and other syndromes (23, 24). The molecular mechanisms that contribute to FGFR gain-of-functions mutations are complex and include constitutive (ligand-independent) or ligand-dependent FGFR activity (2528). In Apert syndrome, excess FGFR2 signaling causes premature suture closure, which is linked to an increase in the rate of osteoblast differentiation and osteogenesis in the subperiosteal region of the bone (29). Analyses of mice with the same FGFR2 mutation exhibited a variable phenotype (30) that was initially difficult to reconcile with the human syndrome (31). However, additional studies in mice indicated that activated FGFR2 promotes maturation of osteoblasts (3234), which is consistent with observations in Apert craniosynostosis (35). Thus, excess FGFR2 activity leads to a common phenotype in mice and humans that is characterized by premature cranial suture ossification (bone deposition).

Genetic and functional studies have revealed some of the signaling pathways that are involved in the development of cranial dysplasias induced by excess FGFR signaling. In the mouse, ERK1/2 activation plays a pathogenic role in craniosynostosis induced by FGFR2 mutations (36, 37). In human craniosynostosis caused by activating mutations in FGFR2, activation of PLCγ and PKCα increase osteoblast gene expression in osteoblast precursor cells, leading to premature osteoblast differentiation (38, 39). In these cells, FGFR2 mutations trigger Cbl-mediated ubiquitin-mediated degradation of two Src family members, Lyn and Fyn, increasing osteoblast differentiation (40, 41). In a mouse model of Crouzon craniosynostosis, increased binding of activated FGFR2 to the adaptor protein FRS2α (42) was also shown to be involved in the negative feedback mechanism induced by FGFR stimulation (43). Thus, several signaling mechanisms activated by FGFR2 are likely to contribute to craniosynostosis, and many of these may converge to regulate the expression of genes involved in cell differentiation (Fig. 1).

Fig. 1

Pathways downstream of FGFR2 implicated in craniosynostosis. Only signaling molecules implicated in craniosynostosis are shown. Not all molecules involved in FGFR signaling are shown.

Credit: Y. Hammond/Science Signaling

An essential transcription factor involved in osteoblastogenesis is Runx2, which is encoded by a gene that is stimulated by FGFR2 signaling (4446). In mice, loss of function of FGFR2IIIc—the mesenchymal splice variant of FGFR2—decreases the transcription of Runx2 and so retards ossification (47). FGFR signaling also represses the expression of other genes. For example, activating FGFR2 mutations results in the decreased expression of some WNT target genes in mice (48, 49). Thus, multiple signaling pathways contribute to the role of FGFR, and FGFR signaling intersects with other developmentally important pathways to control osteoblast differentiation and skeletogenesis.

Crosstalk Between FGFR and Other Pathways in Skeletogenesis

Crosstalk between FGFR and WNT and BMP pathways

Crosstalk between the FGFR pathway and of other signaling cascades controls mesenchymal stem cell fate and differentiation in normal bone development and contributes to craniosynostosis. WNT-β-catenin signaling is also an important regulator of osteogenesis. WNT-β-catenin signaling increases bone mass through a number of mechanisms, including stimulation of the renewal of stem cells, stimulation of preosteoblast replication, and induction of osteoblastogenesis (13). Loss-of-function mutations in human LRP5 (WNT co-receptor, low-density lipoprotein receptor–related protein 5) are associated with osteoporosis-pseudoglioma syndrome, which is characterized by low bone-mineral density and skeletal fragility (50).

Studies in mice implicated the WNT-β-catenin pathway in cranial suture closure by showing that knocking out AXIN2, which encodes a negative regulator of WNT signaling, causes premature suture closure through activation of WNT signaling (51, 52). Genetic analyses in mice revealed that the WNT and FGFR pathways interact to control mesenchymal stem cell differentiation, controlling suture closure (53). Simultaneous disruption of AXIN2 (complete loss) and FGFR1 (heterozygous deficiency) increased the differentiation of mesenchymal cells into chondrocytes, causing abnormal suture closure (53). Activated β-catenin signaling directly increases stem cell proliferation and BMP signaling, whereas decreased FGFR1 signaling facilitates chondrocyte differentiation of mesenchymal stem cells (Fig. 2). Thus at least in mice, the balance between FGFR, WNT, and BMP signaling is a determinant of mesenchymal stem cell fate during cranial suture development.

Fig. 2

The balance of FGFR and WNT and BMP signaling controls chondrocyte differentiation. Either loss of function of FGFR or gain of function can result in craniosynostosis through chondrocyte differentiation or osteoblast differentiation, respectively.

Credit: Y. Hammond/Science Signaling

Crosstalk between FGFR and EGFR and PDFGRα pathways

Crosstalk between the FGFR pathway and two other RTKs, EGFR and PDGFRα, has also been implicated in skeletal development and osteoblast dysfunction that leads to craniosynostosis. A transcriptomic analysis revealed that FGFR2 activation induced by gain-of-function FGFR2 mutations elicited the expression and signaling of both the EGFR and PDGFRα in human osteoblasts and in cranial fused sutures from Apert patients. Furthermore, the increased EGFR and PDGFRα signaling induced by aberrant FGFR2 signaling appears to be functionally involved in the abnormal osteoblast phenotype in this skeletal disorder (54). Mechanistic studies revealed that FGFR2 stimulates the expression of the genes encoding the EGFR and PDGFRα through PKCα-mediated AP-1 transcriptional activation (Fig. 3). In addition to this transcriptional action, FGFR2 activity controls EGFR abundance by a posttranscriptional mechanism involving Sprouty2 (Spry2). Spry2 is a docking protein that is generally considered to be an inhibitor of receptor tyrosine kinases (55). However, Spry2 can also potentiate EGFR signaling by specifically intercepting Cbl-mediated effects on receptor down-regulation (56). Upon stimulation of FGFR or EGFR, phosphorylation of Spry2 in the presence of FRS2α results in interaction with Cbl, which recruits this ubiquitin ligase to the activated receptor and promotes receptor ubiquitination and degradation (57, 58). In osteoblasts with FGFR2 with activating mutations, the Cbl-Spry2 interaction occurs and sequesters Cbl away from the EGFR, impeding EGFR ubiquitination and degradation and increasing EGFR abundance and signaling (Fig. 3) Thus, several mechanisms appear to be involved in the up-regulation of EGFR and PDGFRα signaling induced by activated FGFR2

Fig. 3

Transcriptional and posttranscriptional crosstalk between activated FGFR2, EGFR, and PDGFRα signaling in osteoblast differentiation. (A) Under normal conditions, ligand binding triggers the Cbl-mediated decrease in EGFR, PDFGRα, and FGFR activity by stimulating receptor internalization and degradation. (B) Activated FGFR2 induced by gain-of-function mutations in humans increase through a posttranscriptional mechanism, involving reduced EGFR degradation as a result of Sprouty2 activation and sequestration of Cbl. The increased EGFR and PDGFRα signaling induced by activated FGFR2 in turn further stimulates PKCα activity, functionally contributing to enhanced osteoblast differentiation and osteogenesis in craniosynostosis. Activating FGFR mutations also increases EGFR and PDGFRα abundance and signaling by a transcriptional mechanism involving PKCα-mediated AP-1 transcriptional activation.

Credit: Y. Hammond/Science Signaling

Potential Therapeutic Implications

In addition to expanding our knowledge of the FGFR signaling processes that regulate skeletogenesis, the finding that crosstalk between the FGFR pathway and other signaling pathways in the control of mesenchymal stem cell fate determination and skeletogenesis may have potential therapeutic implications. The observations that antagonizing FGFR signaling can prevent skeletal dysplasias induced by FGFR mutations (59) and that attenuation of FGFR signaling by pharmacological intervention prevents craniosynostosis in mice (37, 42, 60) led to the promising concept that antagonists of FGFR signaling may prevent cranial dysplasias induced by aberrant FGFR signaling. However, FGFR signaling is important for many developmental processes, and globally inhibiting FGFR signaling may have undesirable consequences. The crosstalk between FGF signaling and other signaling pathways that control mesenchymal stem cell fate determination and skeletogenesis may offer novel therapeutic strategies in FGFR-related skeletal disorders. Notably, attenuation of signaling by drugs that inhibit EGFR or PDGFRα may correct the abnormal osteoblast phenotype and premature suture fusion in severe skeletal dysplasia linked to activating FGFR mutations and, perhaps, in other human disorders characterized by aberrant FGFR signaling. Further investigation of the functional interplay between FGFR and other signaling pathways will reveal whether these pathways cooperate to elicit the cellular responses to activated FGFR signaling in other pathological contexts, notably in cancer.

References

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