[Frontiers in Bioscience 3, d781 -794, August 1, 1998]
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FGF SIGNALING IN SKELETAL DEVELOPMENT

Michael C. Naski and David M. Ornitz

Department of Molecular Biology and Pharmacology, Washington University School of Medicine, Campus Box 8103, 660 S. Euclid Ave., St. Louis, MO 63110

Received12/22/98 Accepted 2/17/98

2. INTRODUCTION

The fibroblast growth factors (FGFs) comprise a family of at least fifteen structurally related proteins (reviewed in (1-5). FGFs were first described in the 1970s as an activity that stimulated the proliferation of NIH 3T3 cells. Since that time, FGFs have been shown to support the proliferation of a variety of both mesenchymal and epithelial cells (2). In addition, FGFs are potent regulators of cell migration (6) and differentiation (2). Animal studies have proven that FGFs are required for diverse developmental processes, including inner ear and tail development (7), hair follicle maturation (8, 9), early embryogenesis (10) and skeletal growth and differentiation (11-13).

The FGF receptors are a family of four receptor tyrosine kinases encoded by four distinct genes (14-21). Each receptor recognizes a unique subset of the FGF family of ligands (22). Alternative splicing of the extracellular domain expands the repertoire of ligands that can be recognized by each receptor, while restricting the repertoire of ligands recognized in specific tissues (23-25). The patterns of receptor expression and alternative splicing match ligand binding specificity with proximally expressed ligands (26, 27). During embryonic development, receptor-ligand pairs are often matched across epithelial-mesenchymal boundaries. This permits the inductive effects FGFs to be targeted to neighboring tissues and thereby regionally instruct cells to proliferate, migrate or differentiate. Receptor-ligand interactions are further modified by heparan sulfate proteoglycans (HSPG). These molecules have been shown by a variety of studies to be required for ligand binding in vitro (20, 28, 29) and appear to be required for the formation of an active receptor complex. This hierarchy of regulation indicates that the specific ligand-receptor interactions of a given cell are determined by i) expression of one or multiple FGF receptors, ii) alternative splicing of the receptor mRNA iii) interactions of the receptor and ligand with heparan sulfate proteoglycans and iv) the ligands present in the cellular environment.

2.1. FGF receptor alternative splicing

FGF receptors are members of the receptor tyrosine kinase superfamily (30). These proteins consist of an extracellular ligand binding domain, a single transmembrane domain and an intracellular tyrosine kinase domain. The FGF receptor extracellular region contains three immunoglobulin-like (Ig-like) domains, a heparin binding domain and a stretch of seven conserved acidic amino acids (figure 1) (14). A hierarchy of alternative mRNA splicing events determine the number of Ig-like folds in the extracellular domain, as well as the ligand binding properties of the receptor. One major splicing event excises the exons encoding the amino-terminal Ig-like domain (domain I) leading to a form of the receptor with two Ig-like domains (figure 1) (31). The ligand binding properties of both the full length (three Ig-like domain) and truncated (two Ig-like domain) receptor appear to be similar (32). Additional RNA splicing events regulate the use of two mutually exclusive exons and result in two alternative versions of Ig-like domain III (IIIb or IIIc) (23, 24, 31). The genomic DNA encompassing the carboxy-terminal half of Ig-like domain III in FGF receptors 1, 2 and 3 is remarkably conserved both in the number of exons and the arrangement of the intron/exon boundaries (23, 33-35). Alternative splicing of these exons results in either IIIb or IIIc isoforms of the FGF receptor (figure 2), dramatically effecting the ligand-receptor binding specificity (23, 25). Alternative splicing is regulated in a tissue-specific manner (36-38). Utilization of either the "b" or "c" exon is dependent upon the identity of the cell which synthesizes the mRNA. The "b" exon appears to be expressed in epithelial lineages while the "c" exon is expressed in mesenchymal lineages (35, 36, 38, 39).

Figure 1. The primary structure of FGF receptors. Top: Full-length FGF receptor with three Ig-like domains. The major alternative splicing pathways will express either Ig-like domain IIIb or IIIc. The stippled region beginning in Ig-like domain III is the sequence subject to alternative splicing. Bottom: Short form of the receptor expressing Ig-like domain II and III. SP, signal peptide; A, acidic region; I, II, III, Ig-like domains; TM, transmembrane domain; KI, kinase insert; P, putative site of autophosphorylation; s-s, disulfide bond.

Figure 2. Alternative splicing of FGF receptors in the immunoglobulin-like domain III region. Alternatively spliced exons IIIa, IIIb, IIIc are shown. Abbreviations as in figure 1.

2.2. Glycosaminoglycan interactions

FGFs bind the glycosaminoglycans, heparin and heparan sulfate (1, 40). Heparan sulfate proteoglycans (HSPGs) are located on the cell surface and within the extracellular matrix and serve as "low affinity" (Kd≈10-9M), high capacity binding sites for FGF (40). The interaction of FGF with HSPGs has been established by demonstrating decreased binding of FGFs to cells deficient in cell-surface heparan sulfate (20, 41, 42). Additionally, treating cells with heparin degrading enzymes or with inhibitors of glycosaminoglycan sulfation inhibits the binding of and response to FGFs (29, 43). The affinity of FGFs for heparin-like molecules may significantly limit the diffusion and release of growth factor into interstitial spaces (40, 44). FGFs may therefore exert their effects very close to their site of production, making the spatial and temporal patterns of expression of FGFs and FGF receptors an important biological regulatory mechanism.

Heparin and heparan sulfate proteoglycans bind directly to FGFs and FGF receptors and thereby modulate the activation of the FGF receptor. Cells that express FGF receptor 1 and are deficient in HSPGs, require heparin in the binding media for high affinity FGF binding (Kd≈2-20x10-11M) (20, 29). Furthermore, heparin is required for FGF to bind to a soluble FGF receptor in a cell-free system and for FGF to activate its receptor when expressed in growth factor (interleukin 3) dependent lymphoid cell lines (22, 23, 28, 45). The mechanism by which FGF interacts with its receptor may involve the formation of a low affinity complex between FGF and the FGF receptor which can then be stabilized by heparin. The increase in affinity between FGF and the FGF receptor in the presence of heparin is estimated to be approximately 4-10 fold (46, 47).

2.3. FGF receptor Mutations in Human Disease

The essential role for FGF receptor signaling in the regulation of skeletal development has been accentuated by studies of human genetic diseases. Recently, several human skeletal dysplasias have been linked to point mutations in the genes encoding FGF receptors 1, 2 and 3 (figure 3). These disorders can be broadly classified into two groups: 1) the dwarfing chondrodysplasias, including hypochondroplasia (HCH) (48), achondroplasia (ACH) (49-52), thanatophoric dysplasia (TD) (53-56), and 2) the craniosynostoses, including Crouzon syndrome (CS) (57-67), Pfeiffer syndrome (PS) (58, 60, 64, 68, 69), Jackson-Weiss syndrome (JWS) (59, 60, 63), Beare-Stevenson cutis gyrata (70), Apert syndrome (AS) (71) and a nonsyndromic craniosynostosis (72). All of the mutations are autosomal dominant and frequently arise sporadically. The great majority of these disorders result from point mutations in the coding sequence of the receptor that result in a single amino acid substitution (figure 3).

Figure 3. Mutations in the FGF receptor genes in human skeletal diseases. Top: FGF receptor 1 showing a single point mutation causing Pfeiffer syndrome (PS). Abbreviations as in Figure 1. Middle: FGF receptor 2 showing the mutations responsible for Crouzon syndrome (CS), Jackson-Weiss syndrome (JWS), Pfeiffer syndrome (PS), Apert syndrome (AS) and Beare-Stevenson cutis gyrata (BS). Bottom: FGF receptor 3 showing the mutations responsible for achondroplasia (ACH), thanatophoric dysplasia (TD), hypochondroplasia (HCH), Crouzon syndrome & acanthosis nigricans (CSAN) and a non-syndromic craniosynostosis (NSC). The stippled line attached to the end of FGF receptor 3 represents an extension of the protein resulting from mutations in the stop codon of the receptor. The numbers represent the position of the amino acid in the coding sequence for the human receptor. Amino acids are abbreviated using standard single letter abbreviations.

The dwarfing conditions, HCH, ACH and TD result from dominant mutations in the FGF receptor 3 gene. HCH is a mild and relatively common skeletal disorder with clinical features similar to that of ACH. ACH is the most common form of genetic dwarfism. ACH is characterized by shortening of the proximal and, to a lesser extent, distal long bones. The cranium of ACH patients is characterized by frontal bossing, and the face is characterized by a depressed nasal bridge. Rare homozygous cases of ACH usually result in neonatal lethality (73). These individuals have features similar to that of TD. TD results from several dominant mutations in the FGF receptor 3 gene. TD is the most common lethal-neonatal skeletal disorder and is clinically similar to homozygous ACH (73).

PS, CS, JWS, BS and AS are clinically distinct syndromes characterized by craniosynostosis (premature closure of the cranial sutures) and distinct facial features. In addition, PS, JWS, BS and AS have variable phenotypes in the distal extremities consisting of syndactyly of the hands and feet or broad thumbs and big toes. In general, the mutations affecting craniofacial development have resulted from mutations in FGF receptors 1 or 2. Recently however,

mutations in FGF receptor 3 have also been shown to cause CS and a non-syndromic craniosynostosis (66, 72) (for review of the FGF receptor mutations and the corresponding clinical abnormalities see (74, 75)).