Control of Bone Growth by Fibroblast Growth Factors

channels: more pieces of the puzzle. Curr. Opin. Cell Biol. 9, 553–559 40 Aguilar-Bryan, L., Clement, J.P.T., Gonzalez, G., Kunjilwar, K., Babenko, A. and Bryan, J. (1998) Toward understanding the assembly and structure of KATP channels. Physiol. Rev. 78, 227–245 41 Clement, J.P.T. et al. (1997) Association and stoichiometry of K(ATP) channel subunits. Neuron 18, 827–838 42 Thomas, P.M. et al. (1996) Inactivation of the first nucleotide-binding fold of the

sulfonylurea receptor, and familial persistent hyperinsulinemic hypoglycemia on infancy. Am. J. Hum. Genet. 59, 510–518 43 Nestorowicz, A. et al. (1996) Mutations in the sulfonylurea receptor gene are associated with familial hyperinsulinism in Ashkenazi Jews. Hum. Mol. Genet. 5, 1813–1822 44 Miki, T. et al. (1997) Abnormalities of pancreatic islets by targeted expression of a dominant-negative Katp channel. Proc. Natl. Acad. Sci. U. S. A. 94, 11969–11973

Control of Bone Growth by Fibroblast Growth Factors Francesco De Luca and Jeffrey Baron Fibroblast growth factors (FGFs) and their receptors (FGFRs) negatively regulate longitudinal bone growth. Activating FGFR3 mutations impair growth, causing human skeletal dysplasias, whereas inactivating mutations stimulate growth. Systemic administration of FGF-2 to mice stimulates bone growth at low doses but inhibits growth at high doses. In organ culture, FGF-2 inhibits growth by decreasing growth plate chondrocyte proliferation, hypertrophy and cartilage matrix synthesis. Local FGF-2 infusion accelerates ossification of growth plate cartilage. Thus, FGFs may regulate both growth plate chondrogenesis and ossification. Longitudinal bone growth occurs at the growth plate. The cartilaginous growth plate is organized into three functionally and structurally distinct layers: the resting, the proliferative and the hypertrophic zones1. The resting zone lies nearest to the epiphysis. The chondrocytes in this zone are irregularly arranged in a bed of cartilage matrix, and they rarely divide. Nearer the metaphysis, in the proliferative zone, the chondrocytes show a flattened shape and are arranged in columns oriented parallel to the long

F. De Luca is at the Division of Pediatric Endocrinology, University of Maryland School of Medicine, Baltimore, MD 21202; J. Baron is at the Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

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axis of the bone. When proliferative zone chondrocytes divide, the daughter cells line up along the long axis of the bone. Thus, the cell columns represent clones of chondrocytes2. The proliferative chondrocytes furthest from the epiphysis stop replicating and instead enlarge to become hypertrophic chondrocytes. These terminally differentiated cells form a layer adjacent to the metaphysis termed the hypertrophic zone3. Longitudinal bone growth occurs by endochondral ossification, a two-step process in which cartilage is first formed and then remodeled into bone. Growth plate chondrocyte proliferation, hypertrophy and extracellular matrix secretion lead to formation of new cartilage, chondrogenesis. Simultaneously, the growth plate is invaded from the metaphysis by blood vessels

45 Weinzimer, S.A., Stanley, C.A., Berry, G.T., Yudkoff, M., Tuchman, M. and Thornton, P.S. (1997) A syndrome of congenital hyperinsulinism and hyperammonemia. J. Pediatr. 130, 661–664 46 Zammarchi, E., Filippi, L., Novembre, E. and Donati, M.A. (1996) Biochemical evaluation of a patient with a familial form of leucine-sensitive hypoglycemia and concomitant hyperammonemia. Metabolism 45, 957–960 47 Ryan, F.D. et al. (1998) Hyperinsulinism: the molecular aetiology of focal disease. Arch. Dis. Child. 79, 445–447

and bone cell precursors, which remodel the cartilage into bone4. These two processes, chondrogenesis and ossification, are tightly coupled so that the width of the growth plate remains relatively constant while new bone is formed at the junction of the growth plate and the metaphyseal bone. The rate of bone growth is determined by the rate of chondrogenesis. During ossification, there is thought to be little change in the physical dimensions of the remodeled tissue. The rate of longitudinal bone growth is regulated by multiple endocrine factors, including growth hormone, insulin-like growth factor I (IGF-I), thyroid hormone, glucocorticoids and sex steroids. In addition, the underlying cellular processes of proliferation, terminal differentiation, angiogenesis and ossification appear to be regulated by a system of paracrine factors, including IGFs, parathyroid hormone-related protein, transforming growth factor β and fibroblast growth factors (FGFs). For FGFs, in particular, there is now strong evidence, both in vitro and in vivo, that members of this growth factor family play a key role in the regulation of endochondral bone formation at the growth plate. •

FGFs and their Receptors

The family of FGFs includes at least 13 known polypeptides, which share up to 55% sequence identity at the amino acid level and are highly conserved among species5. FGFs have been implicated in many physiological and pathological processes, such as embryonic development, neuronal outgrowth, cell

1043-2760/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S1043-2760(98)00120-9

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Growth rate (µm day–1)

150

100

*

50

0 0

1000 FGF-2 (ng ml–1)

Figure 1. Longitudinal growth rate (mean ±SEM) of fetal rat metatarsals (embryonic day 20) cultured for two days in serum-free medium containing 0 or 1000 ng ml–1 FGF-2. *p <0.001.

survival, angiogenesis and malignant transformation. FGFs evoke a cellular response by interacting with high-affinity transmembrane receptors6. Four FGF receptors (FGFRs) have been identified to date. Their structure includes an extracellular domain, a transmembrane domain and a cytoplasmic tyrosine kinase domain. The four FGFRs are highly homologous, with ~80% amino acid identity in the ligand-binding domain and in the tyrosine kinase domain. The extracellular portion, which consists of two or three immunoglobulin (Ig)-like loops, interacts with the FGF ligand. The C-terminal half of Iglike domain III is encoded by three alternatively spliced exons: IIIa, IIIb and IIIc. The expression of the alternative exons in this position appears to determine ligand specificity and affinity. Alternative splicing also results in the synthesis of soluble forms of FGFRs. FGFs contain low-affinity binding sites for the heparan sulfate proteoglycans (HSPGs). It is thought that binding of FGFs to HSPGs results in protection and storage of FGFs in the extracellular matrix. HSPGs present on the cell surface may also bind FGFs and appear to be required for the interaction with the highaffinity receptors7. After FGF binding, FGFRs dimerize. Dimerization leads to autophosphorylation and transphosphorylation of the

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cytoplasmic domains of the receptors. Studies of FGFR1 and FGFR3 suggest that FGFs signal through multiple intracellular pathways. The activation of Ras-dependent and STAT signaling pathways results in stimulation and inhibition of mitogenesis, respectively8. FGFRs also induce phospholipase Cγmediated phosphatidyl inositol hydrolysis, although the biological importance of this effect is not known9. Another signaling pathway might involve the internalization and nuclear translocation of liganded FGFRs (Ref. 10). •

Skeletal Expression of FGFs and FGFRs

FGFs and FGFRs are produced in skeletal tissues during embryonic and postnatal life. The production of FGF4 in mice appears to be necessary for limb pattern formation11. FGFR1 and FGFR2 mRNA molecules are both found in prebone and precartilage structures during craniofacial bone formation12. In the growth plate of several animal species, including humans, FGF-1 and FGF-2 are found in the proliferative and upper hypertrophic zone chondrocytes13,14. Three of the high-affinity FGFRs are also found in the growth plate of the mouse embryo15. FGFR1 is produced in the hypertrophic chondrocytes, whereas FGFR2 is present exclusively in the perichondrium and periosteum. FGFR3 is found in the resting and proliferative chondrocytes. The distribution of FGF receptors may differ among mammalian species. In the fetal sheep, FGFR1 is abundant in the proliferative zone and is lost in the hypertrophic zone16. •

Effects of FGFR Mutations on Bone Growth

The identification of FGFR mutations in humans with skeletal dysplasias has contributed greatly to the understanding of the biological role of FGFs and their receptors in skeletal growth. Achondroplasia, the most common form of skeletal dysplasia, is characterized by macrocephaly, lumbar lordosis, narrowing of the spinal column and marked proximal shortening of the extremities. Achondroplasia is

inherited as an autosomal dominant trait, although more than 80% of cases are sporadic. Thanatophoric dysplasia is a more severe skeletal dysplasia. Growth of the long bones is severely affected. The ribs are also short, preventing adequate ventilation. A third skeletal dysplasia, hypochondroplasia, has a phenotype that is similar but milder than that of achondroplasia. All three skeletal dysplasias are caused by abnormal endochondral ossification, which results in impaired growth of the long bones and the base of the skull. In achondroplastic patients, the normal histology of the growth plate is generally preserved, with chondrocytes still arranged in normal columns and rows17. Although the rate of endochondral ossification appears to be reduced in these disorders, membranous ossification (bone formation without a cartilage intermediary) is normal. Because of this differential effect on the modes of ossification, achondroplasia is associated with shortened long bones and relative overgrowth of the periosteum and perichondrium. In achondroplasia, the normal rate of membranous ossification and the quantitative decrease in endochondral ossification also account for a relatively normal cranial vault and a shortened base of the skull, respectively. Mutations in the FGFR3 gene have been identified in individuals with achondroplasia, thanatophoric dysplasia and hypochondroplasia18–20. More than 95% of the patients with achondroplasia studied so far have a heterozygous substitution (G380R) in the transmembrane domain of FGFR3. Similarly, a mutation (N540K) affecting the cytoplasmic domain of FGFR3 has been identified in most of the patients affected with hypochondroplasia. Mutations associated with thanatophoric dysplasia have been recognized in several sites of the extracellular and cytoplasmic domains of FGFR3. In contrast to FGFR3 mutations, which primarily affect long bones, FGFR1 and FGFR2 mutations primarily affect the intramembranous bones of the skull, resulting in four craniosynostosis syndromes, namely: TEM Vol. 10, No. 2, 1999

Apert, Pfeiffer, Jackson-Weiss and Crouzon21–23. Premature fusion of the cranial sutures leads to abnormal skull shape, proptosis and mid-face hypoplasia. Hand and foot anomalies are also characteristic of the Apert, Pfeiffer and Jackson-Weiss syndromes. The sequence alterations associated with these skeletal dysplasias are gainof-function missense mutations. This conclusion is supported by the absence of skeletal abnormalities in individuals with a deletion of 4p, who are lacking one copy of the FGFR3 gene (Wolf-Hirschorn syndrome). Furthermore, FGFR3 mutations associated with skeletal dysplasias have been shown to activate the receptor in vitro8,24. This receptor activation may involve several mechanisms, such as ligand-independent dimerization and increased tyrosine kinase activity. Similar evidence exists that FGFR1 and FGFR2 mutations associated with craniosynostosis syndromes also cause receptor activation25. Inactivating FGFR mutations have not yet been detected in humans. However, in mice, inactivating mutations have been introduced into the FGFR3 gene26,27. Mice that are homozygous for these FGFR3 mutations show overgrowth of the long bones and vertebrae, indicating enhanced endochondral bone formation. Thus, activating FGFR3 mutations inhibit longitudinal bone growth, whereas inactivating FGFR3 mutations stimulate longitudinal bone growth. These findings suggest that FGFR3 normally restrains chondrogenesis in the mammalian growth plate, thus inhibiting bone growth. •

Effects of FGFs on Growth Plate Chondrogenesis

FGF-2 (basic FGF) is a potent ligand for FGFR3. It is an 18-kDa, 155-amino acid protein5. In vertebrates, FGF-2 is found in most tissues examined, including the growth plate13,14. Several lines of evidence suggest an involvement of FGF-2 in the regulation of skeletal growth. Transgenic mice overexpressing the gene encoding FGF-2 show long bones considerably shorter than those of TEM Vol. 10, No. 2, 1999

Figure 2. Photomicrograph of rabbit tibial growth plate receiving FGF-2 infusion. FGF-2 was infused for six days with an osmotic minipump attached to a fine needle. The needle was inserted into the epiphyseal bone immediately adjacent to the proximal tibial growth plate of sixweek-old rabbits. Infusion of vehicle into the contralateral tibia did not cause any acceleration of vascular invasion or ossification. A, area of induced vascular invasion; E, epiphyseal bone; G, growth plate; M, metaphyseal bone; N, path of infusion needle. Reproduced with permission from Ref. 41.

wild-type animals28. The growth plates from these transgenic mice show enlargement of the reserve and proliferative zones, while the size of the hypertrophic zone is greatly reduced. The vertebrae appear to be shortened and deformed and the ribs wide and flattened. These transgenic mice also exhibit macrocephaly. Systemic administration of FGF-2 in growing rats has a biphasic effect on longitudinal bone growth29. The intravenous injection of 0.1 mg kg–1 day–1 FGF-2 results in an increase in longitudinal bone growth and cartilage cell proliferation. By contrast, a higher dose of FGF-2 (0.3 mg kg–1 day–1) induces a significant reduction in the longitudinal growth rate and the cartilage cell proliferation rate. The rate of longitudinal bone growth depends on the rate of growth plate chondrogenesis. Therefore, FGF-2 could be slowing bone growth by inhibiting one of the cellular processes underlying chondrogenesis, namely: chondrocyte proliferation, chondrocyte

hypertrophy and cartilage matrix synthesis. To determine which of these processes is regulated by FGF-2, several groups have studied isolated growth plate chondrocytes in cell culture. In vitro, FGF-2 inhibits terminal differentiation and hypertrophy of growth plate chondrocytes. As cultured chondrocytes undergo terminal differentiation, the number of highaffinity binding sites for FGF-2 decreases30. This finding suggests that a decrease in FGFR may also cause chondrocyte terminal differentiation and hypertrophy in vivo. In most reported studies, FGF-2 acts as a potent mitogen for growth plate chondrocytes. Trippel et al.31 have demonstrated that FGF-2 stimulates thymidine incorporation in cultured chondrocytes isolated from bovine and rat growth plates. Similar results have been reported in chondrocytes grown from other animal species32–34. However, Makower et al.35 found that FGF-2 decreased thymidine 63

incorporation in cultured rat growth plate chondrocytes. Similarly, discordant results have been reported concerning the effects of FGF-2 on matrix synthesis in cultured growth plate chondrocytes. FGF2 has been shown to stimulate both collagen and glycosaminoglycan synthesis in chondrocytes isolated from the embryonic chick36, ovine fetus37 and postnatal rabbit38. By contrast, Horton et al.39 showed that FGF-2 suppressed type-II collagen synthesis by embryonic chick chondrocytes. Thus, studies of the effects of FGF-2 in isolated chondrocytes in culture have produced results that are inconsistent and do not readily explain the effects of FGF-2 in vivo. A likely explanation for these discrepancies is that the precise response of the cells might depend on the specific conditions in the culture dish. In a number of carefully studied systems, it has been shown that the effects of growth factors on growth plate chondrocytes can be highly dependent on concentration, presence of other growth factors, growth conditions and other variables40. Furthermore, a cell culture system does not maintain the intercellular and cell–matrix interactions present in the highly organized growth plate. The use of an organ culture system circumvents many of the limitations of isolated cell culture. The effects of FGF-2 have been studied in rat fetal metatarsals maintained in serum-free medium. In this system, addition of FGF-2 to the culture medium inhibits longitudinal growth, a finding consistent with the effects in vivo41 (Fig. 1). In this system, FGF-2 produced a puzzling increase in the overall rate of DNA synthesis, similar to that observed in isolated cell culture. However, autoradiographic studies showed that this increase occurred exclusively in the perichondrium, a structure that does not contribute greatly to the rate of longitudinal bone growth. In the epiphyseal and proliferative chondrocytes, the proliferation rate was decreased. These findings demonstrate the importance of studying specific cell populations that maintain their

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normal spatial relationships in the growth plate. FGF-2 also decreased chondrocyte hypertrophy, a finding consistent with observations made in cell culture. •

Effects of FGF-2 on Growth Plate Ossification

Newly formed cartilage in the growth plate is remodeled into bone. The hypertrophic zone of the growth plate is invaded by metaphyseal capillaries. Bone cells follow the vascular invasion, causing removal of cartilage and replacement by bone. Vascular invasion and ossification appear to be induced by a signal (or signals) from the hypertrophic zone42. Although the nature of this signal has not been firmly established, there is some evidence that FGFs play a role. Direct infusion of FGF-2 into the rabbit tibial growth plate accelerates vascular invasion, cartilage remodeling and ossification of the growth plate43 (Fig. 2). Although the effects elicited by a single high concentration of FGF-2 might not reflect the biological actions of endogenous FGF-2, they suggest that FGF-2 might play a physiological role in the regulation of angiogenesis and ossification in the growth plate. Increased vascular invasion was not seen in transgenic mice overexpressing the gene encoding FGF-2. One possible explanation for this discrepancy might be that, in the FGF-2 transgenic mice, there may be increased synthesis of FGF-2 throughout the growth plate and bone. By contrast, local infusion of FGF-2 into the epiphyseal margin of the growth plate might lead to a decreasing concentration gradient from the resting zone towards the hypertrophic zone. A similar but weaker gradient, which is present physiologically in the growth plate44, might be responsible for attracting the normal vascular invasion from the metaphyseal bone. •

Conclusions

Fibroblast growth factor receptors FGFR1, -2 and -3 are expressed in the growth plate. Gain-of-function mutations in these receptors cause skeletal

abnormalities in humans. Mutations in FGFR3 in particular inhibit longitudinal bone growth in hypochondroplasia, achondroplasia and thanatophoric dysplasia. Conversely, inactivating mutations in FGFR3 stimulate longitudinal bone growth in mice. Thus, FGFR3 appears to restrain longitudinal bone growth at the growth plate. Two of the ligands for these receptors, FGF-1 and FGF-2, are also found in the growth plate. Overexpression of FGF-2 in mice inhibits bone growth. Systemic administration of FGF-2 has a biphasic effect on growth, showing stimulation at lower doses and inhibition at higher doses. Organ culture studies suggest that the growth inhibition caused by high concentrations of FGF-2 might be mediated by three cellular mechanisms: decreased chondrocyte proliferation, decreased chondrocyte hypertrophy and decreased cartilage matrix synthesis. Direct infusion of FGF-2 into the rabbit growth plate accelerates vascular invasion and ossification of growth plate cartilage, suggesting that endogenous FGFs might not only regulate chondrogenesis, but might also participate in the regulation of the ossification of the newly formed cartilage. References 1 Howell, D.S. and Dean, D.D. (1992) The biology, chemistry, and biochemistry of the mammalian growth plate. In Disorders of Bone and Mineral Metabolism (Coe, F.L. and Favus, M.J., eds), pp 313–353, Raven Press 2 Hunziker, E.B. (1994) Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes. Microsc. Res. Tech. 28, 505–519 3 Farnum, C.E. and Wilsman, N.J. (1987) Morphologic stages of the terminal hypertrophic chondrocyte of growth plate cartilage. Anat. Rec. 219, 221–232 4 Aharinejad, S. et al. (1995) Microvascular pattern in the metaphysis during bone growth. Anat. Rec. 242, 111–122 5 Bikfalvi, A., Klein, S., Pintucci, G. and Rifkin, D.B. (1997) Biological roles of fibroblast growth factor-2. Endocr. Rev. 18, 26–45 6 Burke, D. et al. (1998) Fibroblast growth factor receptors: lessons from the genes. Trends Biochem. Sci. 23, 59–62 7 Klagsbrun, M. and Baird, A. (1991) A dual receptor system is required for basic fibroblast growth factor activity. Cell 67, 229–231

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35 Makower, A.M., Wrobleski, J. and Pawlowski, A. (1989) Effects of IGF-I, rGH, FGF, EGF, and NCS on DNA synthesis, cell proliferation and morphology of chondrocytes isolated from rat rib growth cartilage. Cell Biol. Int. Rep. 13, 259–270 36 Kato, Y., Iwamoto, M. and Koike, T. (1987) Fibroblast growth factor stimulates colony formation of differential chondrocytes in soft agar. J. Cell. Physiol. 133, 491–498 37 Hill, D.J., Logan, A., McGarry, M. and De Sousa, D. (1992) Control of protein and matrix molecular synthesis in isolated ovine fetal growth plate chondrocytes by the interactions of basic fibroblast growth factor, insulin-like growth factors-I and -II, insulin and transforming growth factor-b. J. Endocrinol. 133, 363–373 38 Inoue, H., Kato, Y., Iwamoto, M., Hiraki, Y., Sakuda, M. and Suzuki, F. (1989) Stimulation of cartilage matrix proteoglycan synthesis by morphologically transformed chondrocytes grown in the presence of fibroblast growth factor and transforming growth factor b. J. Cell. Physiol. 138, 329–337 39 Horton, W.E., Higginbotham, J.D. and Chandrasekhar, S. (1989) Transforming growth factor-beta and fibroblast growth factor act synergistically to inhibit collagen II synthesis through a mechanism involving regulatory DNA sequences. J. Cell. Physiol. 141, 8–15 40 Hill, D.J. and Logan, A. (1992) Peptide growth factors and their interactions during chondrogenesis. Prog. Growth Factor Res. 4, 45–68 41 Mancilla, E.E., De Luca, F., Uyeda, J.A., Czerwiec, F.S. and Baron, J. (1998) Effects of fibroblast growth factor-2 on longitudinal bone growth. Endocrinology 139, 2900–2904 42 Abad, V., Uyeda, J.A., Temple, H.T., De Luca, F. and Baron, J. Determinants of spatial polarity in the growth plate. Endocrinology (in press) 43 Baron, J. et al. (1994) Induction of growth plate cartilage ossification by basic fibroblast growth factor. Endocrinology 135, 2790–2793 44 Sasse, J., Sullivan, R. and Klagsburn, M. (1987) Purification of cartilage-derived growth factors. Methods Enzymol. 146, 320–325

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