FGFR3-related dwarfism and cell signaling
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KeywordsFibroblast growth factor receptor 3 Cell signaling Dwarfism Clinical therapy
Skeletal development consists of the following steps: skeletal patterning, mesenchymal differentiation, bone growth, and homeostasis. In the early phase of embryogenesis, immature mesenchymal cells gather in the proper position, and the anteroposterior and dorsoventral axes are determined. This “skeletal patterning” is followed by “differentiation” of immature mesenchymal cells to chondrocytes or osteoblasts. In the appendicular skeleton, endochondral ossification takes part in elongation, and chondrocytes create the growth plate, a chondrocytic layer, and proliferate with high frequency in the longitudinal direction. Bone growth continues until the growth plate is closed at the time of pubertal maturation. In mature bones, a homeostasis process called bone remodeling occurs. Bone remodeling always renews old bone tissue by forming both osteoblasts and resorbing osteoclasts.
Many essential genetic mechanisms for these bone-growing steps have been described. Disorders of each bone formation step cause characteristic skeletal dysplasias. For example, polydactyly and split-hand are caused by patterning failure, dwarfism is caused by extraordinary differentiation and proliferation of chondrocytes in the growth plate, and osteoporosis and osteopetrosis are caused by abnormality of bone remodeling. Many functional genes are identified through genetic disorders of the skeleton and knockout mice. Skeletal disorders have been classified based on the responsible genes , clinical features , and molecular pathology and embryology . According to the reviews, a total of 271 clinically different disorders and 75 responsible genes have been described. The most frequent genetic skeletal dysplasia is achondroplasia (ACH) (OMIM; #100800), caused by point mutations in the fibroblast growth factor receptor 3 (FGFR3; OMIM *134934) gene [4, 5]. Some other skeletal dysplasias are also caused by mutations located in the FGFR3, such as hypochondroplasia (HCH) (OMIM; #146000), thanatophoric dysplasia type I (TDI) (OMIM; #187600) and type II (TDII) (OMIM; #187601), and severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN) (description in OMIM *134934.0015) [6, 7, 8, 9, 10, 11]. The location of each mutation is unique. The phenotypes of those FGFR3-related skeletal dysplasias are similar to that of ACH, but the severity is quite different. Mutations of the FGFR3 gene have been reported to be responsible for craniofacial disorders and multiple myeloma, which are not a focus in this review. Many reports state all mutant FGFR3s are constitutively activated. Excessive phosphorylation of FGFR3 triggers intracellular signaling within the chondrocytes of the growth plate to terminate its proliferation .
In this review, we describe the correlation between mutations in the FGFR3 gene and clinical symptoms focusing on the molecular mechanism of mutated FGFR3, and we classify FGFR3-related skeletal dysplasias based on clinical severity and the essential molecular mechanism of mutated FGFR3. Furthermore, we describe the known signaling pathways and try to suggest therapeutic possibilities.
Fibroblast growth factor receptor 3 (FGFR3) and its mutations
Each mutation of FGFR3 (see Fig. 1) for FGFR3-related skeletal dysplasias is a gain-of-function type so that FGFR3 mutants, although the degree is different, stimulate excessive intracellular signaling [21, 22]. Activation of FGFR3 leads to permanent phosphorylation of the receptor itself, so that chondrocytes in the growth plate differentiate excessively and terminate through the intracellular signaling pathways.
The point mutations for ACH are G375C and G380R, both in the TM domain of FGFR3 (see Fig. 1). This mechanism of uniformity, tight genotype–phenotype correlation, is genetically remarkable. It is also known that the TM domain is the position of the CpG island that easily mutated by methylation . Various mutations for HCH are reported in FGFR3, such as N540K (the most frequent mutation), and I538V in the TK1 domain and K650N in the TK2 domain. Recently, N328I in an extracellular region was found at the N-glycosylation site N–X–T motif [24, 25, 26, 27]. Some HCH patients, however, do not have any mutation of the coding region of FGFR3, implying other causes. Many mutations of FGFR3 for TDI are reported, divided into two groups: changing to cysteine residue in the extracellular domain (R240C, A249C, G370C, Y373C), and stop codon mutations (X807C, X807R, X807G) [28, 29, 30, 31]. On the other hand, the mutation for TDII has been known only in K650E in the TK2 domain [29, 32]. The difference of the mutation pattern indicates that TDI and TDII are quite different diseases. The mutation for SADDAN is found at K650 of FGFR3, the same as TDII (K650E), but to a different amino acid, methionine (K650M) [33, 34]. It is interesting that quite different dysplasias are caused by related mutations of the same codon, K650. Actually, the amino acid range around K650 is called the activation loop, implicated as an important region for FGFR3 function, but the detailed functions have not been sufficiently described.
Clinical features of FGFR3-related dwarfisms
ACH is an autosomal dominant skeletal dysplasia characterized by rhizomelic short limbs and short stature, with the highest incidence of around 1.0 per 10,000 live births [4, 5, 35]. The adult height is 120–140 cm. Characteristic features for clinical diagnosis are a long and narrow trunk, a large head with frontal bossing, hypoplasia of the midface, depressed nasal bridge, and trident hands . Some features other than abnormal shape formation are hypotonia and delayed motor milestones, but intelligence is normal, unless hydrocephalus or other central nervous system complications arise [37, 38].
HCH is characterized by mild short limbs and short stature, and mild mental retardation (9% of the patients), but no abnormality of face or trident hands [6, 7]. The adult height is 140–150 cm. However, some HCH patients do not have any mutations in FGFR3 [6, 39, 40]. Therefore, HCH is not as uniform clinically and genetically as ACH, and there might be other responsible genes.
The incidence of TD is about 1:20,000 among live births [41, 42]. Clinical aspects are characterized by severe rhizomelic short limbs and short stature, macrocephaly with depressed nasal bridge, severe platyspondylisis, and reduced thoracic cavity . Most TD patients die in the perinatal period of dyspnea. TD is classified into two types by the shape of the femur. Femurs of type I TD (TDI) are curved, so-called telephone receiver like, and those of type II TD (TDII) are straight [44, 45]. The characteristic shape of the head of TDII patients is called a cloverleaf skull [44, 46].
SADDAN is used to be called the skeletal-skin-brain syndrome. Although it shows quite severe skeletal anomalies, SADDAN has a totally different clinical phenotype from TDII . SADDAN patients have severe short limbs and short stature, arcuation of tibia and femur, developmental delay, and acanthosis nigricans, but do not have craniosynostosis. In spite of severe osteodysplasty, SADDAN patients are able to survive with respiratory care. Adult height is around 105 cm.
Classification of FGFR3-related dwarfism by clinical severity and by molecular features in vitro
FGFR3 signaling for bone growth
Recent studies imply the existence of signaling crosstalk with endocrine or paracrine signaling molecules, including insulin-like growth factor 1 (IGF-1), C-type natriuretic peptide (CNP), and parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP). IGF-I signaling is an important mediator of growth hormone effects. Because the disturbed appendicular skeleton of ACH is rescued by growth hormone (GH) treatment, for evidence of GH treatment, we proved that IGF-1 signaling prevents chondrogenic cell line ATDC5 cells from expressing activated FGFR3 by apoptosis through the PI3K pathway . CNP induces activation of cyclic guanosine monophosphate (cGMP) through interaction with its receptor, natriuretic peptide receptor B (NPR-B). Both CNP and NPR-B are expressed in the proliferative and prehypertrophic zones of the growth plate, and activation of NPR-B triggers matrix synthesis in chondrocytes, repressing the MAPK pathway downstream of FGFR3 activation . Activation of FGFR3 decrease the expression of bone-forming proteins such as Indian hedgehog (Ihh), Patched (Ptc), and bone morphometric protein 4 (BMP4) . Ihh affects chondrocytes to produce PTHrP. Indeed, expression of PTHrP is decreased in ATDC5 expressing activated FGFR3 .
As therapy for FGFR3-related dwarfisms, including ACH and HCH, treatment with GH has been tried [62, 63, 64]. We also reported that GH treatment for skeletal dysplasias produces a positive effect for a short time . However, GH treatment for ACH does not have as great an effect for GH deficiency, and the effect does not persist very long. Furthermore, excessive GH medication closes the growth plate early, and no clear long-term benefit has been established .
Surgical limb lengthening has been also widely tried to increase the height of affected patients [66, 67, 68]. This method elongates achondroplastic or hypochondroplastic limbs as much as 15–30 cm, but there are many problems, such as the need for repeated surgeries, superficial wound infection, sequelae, and complications .
Investigation of intracellular signaling leads to some clues for the development of novel medications. We demonstrated that one cause of FGFR3-related skeletal dysplasia is apoptosis, responsible for decreased expression of parathyroid hormone-related peptide (PTHrP) . Administration of PTH decreases apoptosis of ATDC5 expressing ACH-FGFR3 or TDII-FGFR3. PTHrP positively affects chondrocyte proliferation in the growth plate through the PTH/PTHrP receptor (PPR). Furthermore, because PPR is the common receptor of both PTH and PTHrP, we investigated possibilities of human recombinant PTH (rhPTH) . Disturbed bone growth of the appendicular skeleton from ACH-FGFR3 transgenic mice was rescued by addition of rhPTH in an organ culture system. This approach is quite realistic, because rhPTH is already established as a medicine for adult osteoporosis.
C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B (NPR-B), are identified as negative regulators and inhibit mitogenesis in rat chondrocytes . Even mutations in the NPR-B gene are responsible for acromesomelic dysplasia . As an in vivo study, overexpression of CNP in chondrocytes has positive effects on ACH-FGFR3 transgenic mice . Because other natriuretic peptides have been used clinically for their hemodynamic effects , CNP or a CNP analogue could be an effective medicine in the future.
Other possible trials were introduced. One is the highly specific antibody against FGFR3 and the other is the selective FGFR3 kinase inhibitor [73, 74]. These approaches have been proven to have a blocking effect on FGFR3 activation in vitro. For development of therapeutic materials, examination of treatment in intact animals is awaited.
From the biological view, FGFs and their receptors comprise various effects on various organs and play a very important role in the skeletal formation. By the genetic approach, each mutation of FGFR3 is quite precisely correlated with the phenotype. It is interesting that point mutations in different positions and/or to different amino acids in the FGFR3 gene result in quite different phenotypes. Although the mechanism of the mutations is complicated, as clues the crucial features are elongated phosphorylation and ER localization of the receptor. However, there might be an unrecognized molecular mechanism for developing mutation-specific phenotypes, such as mental retardation for HCH, and a lethal phenotype for TDI and TDII, but not for SADDAN, by hypoplastic lung.
Among the many genetic rhizomelic dwarfisms, FGFR3-related dwarfisms are the most common disorders. Especially, ACH and HCH patients are able to live out their entire lifetime with many difficulties throughout their social life. We desire to cure those patients; however, there are quite a few radical therapies for congenital disorders. To develop such a therapy, we need to understand the causes and mechanisms of each disease. Therefore, needless to say, clinical follow-up, and genetic or molecular and cellular biological research, are very important.
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