* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project
Achondroplasia: pathogenesis and implications for future treatment Melanie B. Laederich and William A. Horton Research Center, Shriners Hospital for Children, Departments of Cell & Developmental Biology and Molecular & Medical Genetics, Oregon Health & Science University, Portland, Oregon, USA Correspondence to William A. Horton, Research Center, Shriners Hospital for Children, 3101 SW Sam Jackson Park Road, Portland, OR 97239, USA Tel: +1 503 221 1537; fax: +1 503 221 3451; e-mail: [email protected] Current Opinion in Pediatrics 2010, 22:516–523 Purpose of review Although the genetic defect underlying achondroplasia has been known for over a decade, no effective therapies to stimulate bone growth have emerged. Here we review the recent literature and summarize the molecular mechanisms underlying disease pathology and examine their potential as therapeutic targets. Currently used preclinical models are discussed in the context of recent advances with a special focus on C-type natriuretic peptide. Recent findings Research on the mutation in Fibroblast Growth Factor Receptor 3 (FGFR3) that causes achondroplasia suggests that disease results from increased signal transduction from the mutant receptor. Thus, current therapeutic strategies have focused on reducing signals emanating from FGFR3. First-generation therapies directly targeting FGFR3, such as kinase inhibitors and neutralizing antibodies, designed for targeting FGFR3 in cancer, are still in the preclinical phase and have yet to translate into the management of achondroplasia. Counteracting signal transduction pathways downstream of FGFR3 holds promise with the discovery that administration of C-type natriuretic peptide to achondroplastic mice ameliorates their clinical phenotype. However, more research into long-term effectiveness and safety of this strategy is needed. Direct targeting of therapeutic agents to growth plate cartilage may enhance efficacy and minimize side effects of these and future therapies. Summary Current research into the pathogenesis of achondroplasia has expanded our understanding of the mechanisms of FGFR3-induced disease and has increased the number of approaches that we may use to potentially correct it. Further research is needed to validate these approaches in preclinical models of achondroplasia. Keywords achondroplasia, c-type natriuretic peptide, endochondral ossification, FGFR3, Fibroblast Growth Factor Receptor 3, growth plate, targeted therapies Curr Opin Pediatr 22:516–523 ß 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins 1040-8703 Introduction Achondroplasia is by far the most common form of dwarfism in humans and the prototype of the human chondrodysplasias . Its clinical phenotype has been recognized for centuries and the notion that it reflects a disturbance of cartilage-mediated (endochondral) bone growth has existed for about 100 years. However, it was not until mutations of the transmembrane receptor Fibroblast Growth Factor Receptor 3 (FGFR3) were identified in the mid-1990s that the molecular pathogenesis of achondroplasia began to be unraveled. Indeed, considerable progress has been made in elucidating FGFR3 as a key negative regulator of endochondral ossification and in delineating the functional impact of disease-causing mutations on growth plate chondrocyte biology. Potential targets for therapeutic intervention have been identified for achondroplasia, and pilot studies 1040-8703 ß 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins have been carried out in mouse models of achondroplasia. This review will examine recent progress in understanding how FGFR3 mutations interfere with bone growth, the application of this knowledge to preclinical trials in mice and the implications of this research for future therapy of children with achondroplasia. Fibroblast Growth Factor Receptor 3 To understand the rationale behind the therapeutic strategies that have been proposed for achondroplasia, it is necessary to briefly discuss the genetic basis of the condition and the biology of FGFR3. Genetics Achondroplasia is inherited as an autosomal dominant trait. However, mapping its genetic locus was hindered DOI:10.1097/MOP.0b013e32833b7a69 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Achondroplasia: pathogenesis and implications Laederich and Horton 517 by the paucity of multigenerational families needed to establish linkage as most cases arise from a new mutation to nonachondroplastic parents. Nevertheless, it was mapped to the distal short arm of chromosome 4 in 1994 and heterozygous mutations were discovered in FGFR3 shortly afterwards [2–5]. Essentially all patients with the classical features of achondroplasia have the same glycine to arginine substitution at position 380 (G380R), which maps to the transmembrane domain of the receptor. Additional FGFR3 mutations were subsequently detected in thanatophoric dysplasia, hypochondroplasia and other disorders whose clinical phenotypes qualitatively resemble achondroplasia [1,6,7]. Despite an initial debate about the functional consequences of these mutations, analysis of mice in which FGFR3 was knocked out, or in which FGFR3 bearing the achondroplasia mutation was overexpressed, revealed that disease-causing mutations lead to gain of receptor function [8–10]. These observations also established FGFR3 as an important negative regulator of endochondral bone growth. Receptor FGFR3 is one of four high affinity FGF receptors (FGFR1–4) [11–13]. It functions as transmembrane receptor tyrosine kinase possessing an extracellular ligand-binding domain, a transmembrane domain and an intracellular split tyrosine kinase domain (Fig. 1). Alternative splicing of the proximal extracellular domain generates ‘b’ and ‘c’ isoforms, which express in a tissuespecific manner and affect ligand specificity; the c isoform is predominantly expressed in skeletal tissues. Figure 1 FGFR3 signal transduction FGF and heparin binding to the extracellular domain of FGFR3 induce dimerization, kinase activation and transphosphorylation of tyrosine residues, leading to activation of downstream signaling pathways such as STAT. Membrane-bound FRS2 constitutively associates with the intracellular juxtamembrane domain of FGFR3, is phosphorylated by the receptor upon activation and serves as scaffolding platform for signal transduction cascades through the MEK/ERK/p38 pathway. CNP binding to NPR-B induces the generation of the second messenger cGMP, which activates PKG, leading to attenuation of the MEK pathway via RAF. CNP, C-type natriuretic peptide; ERK, extracellular signal-related protein kinase; FGFR3, Fibroblast Growth Factor Receptor 3; FRS2, Fibroblast Growth Factor Substrate 2; NPR-B, natriuretic peptide receptor-B; STAT, signal transducer and activator of transcription. and provides additional tyrosine residues that are phosphorylated in response to receptor activation. Signaling Activation Like other FGFRs, FGFR3 is activated by FGF ligands released from cells near the FGFR3-bearing target cells. These ligands are often referred to as canonical or paracrine FGFs; they include FGF 1–10, 16–18, 20 and 22 . The physiologic ligand(s) for FGFR3 is (are) not known, although FGFs 2, 4, 9 and especially 18 are the best candidates based on their distribution of expression and ability to bind and activate FGFR3, and the phenotypes of mice lacking these ligands [12,15–17]. Heparin or heparan sulfate proteoglycans are required for FGF– FGFR3 interactions and likely influence binding specificity and ligand localization. The current dogma holds that canonical FGFs binding to their cognate FGFRs induce dimerization of receptors, leading to transactivation of the receptors’ tyrosine kinase activity and transphosphorylation of key tyrosine residues within the receptors’ kinase domain [11,18–21]. Signals are propagated by the recruitment of signaling molecules to these phosphorylated residues or to closely linked docking proteins, namely Fibroblast Growth Factor Substrate 2 (FRS2), which binds to the receptor A number of signaling pathways have been detected downstream of FGFR3 activation, including the signal transducer and activator of transcription (STAT), mitogen-activated protein kinase (MAPK), and phospholipase Cg pathways, the first two receiving the most attention (Fig. 1) [11,22–26]. STAT signals are thought to inhibit chondrocyte proliferation, whereas MAPK signals not only negatively influence proliferation but also disrupt terminal differentiation and postmitotic matrix synthesis [24,27–29]. Gene expression studies suggest that FGFR3 signals through multiple pathways that downregulate growth-promoting molecules, leading to reduced proliferation and differentiation of chondrocytic cells in growing bones [30,31]. Consequences of the mutation As noted earlier, the mutations that cause achondroplasia act by exaggerating the negative regulatory functions of FGFR3 on endochondral ossification [1,21,32]. To understand how this occurs, one must consider the impact of the mutation on the receptor itself and that on the growing skeleton. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 518 Endocrinology and metabolism Impact on the receptor The achondroplasia mutation maps to the transmembrane domain of the receptor. It has been proposed that the amino acid substitution stabilizes the receptor dimer, although this finding has been challenged [33,34]. Another mechanism involves delayed turnover of activated receptor, which can lead to an increase in overall signal output. For instance, Monsonego-Ornan  and colleagues have suggested that the achondroplasia mutation slows receptor internalization, leaving it on the surface to signal. Others have described a defect in c-Cbl-mediated receptor ubiquitination that delays trafficking of mutant FGFR3 to lysosomes for degradation [36,37]. Similarly, Ben-Zvi et al.  have suggested that SOCS3 (suppressor of cytokine signaling 3) induced in response to exaggerated STAT1 signals may prolong the survival of mutant FGFR3. Impact on the growing skeleton Endochondral ossification is responsible for both the formation and linear growth of most of the skeleton (Fig. 2). However, the negative regulatory influence of FGFR3 is exerted mainly in the growth phase, in which it reduces the rate of cartilage template formation and turnover necessary for bone elongation. Most evidence to date suggests that FGFR3 inhibits both the proliferation and terminal differentiation of growth plate chondrocytes and synthesis of extracellular matrix by these cells [12,39,40]. This inhibitory function is consistent with its expression in cells exiting the cell cycle. How- ever, it is also proposed that FGFR3 induces premature terminal differentiation, reducing the number of cells that contribute to template synthesis [29,41]. Alteration of downstream signals While all of the pathways that normally propagate FGFR3 signals presumably transmit the exaggerated output of the mutant receptors, there is evidence that the extracellular signal-related protein kinase (ERK) and p38 arms of the MAPK pathway are especially important. Indeed, transgenic mouse strains in which expression of constitutively active MEK1 and MEK6 was targeted to cartilage displayed ‘achondroplastic’ skeletal phenotypes; MEK1 and MEK6 lie upstream of ERK and p38 phosphorylation cascades and constitutively activate these downstream targets [28,42]. Additionally, humans with mutations in proteins involved in the RAS/MAPK pathway that cause constitutive activation of this pathway demonstrate reduced growth and craniosynostosis, further supporting that activation of this pathway plays a role in human skeletal disorders [43,44]. MAPK signals that slow bone growth are subject to downregulation by the signaling cascade activated by C-type natriuretic peptide (CNP) [21,45]. Indeed, genetic manipulation of this pathway up or down in mice leads to skeletal overgrowth or undergrowth, respectively; and overexpression of CNP ameliorates dwarfism in achondroplastic mutant mice as discussed in more detail below [45,46]. Figure 2 Architecture and zones of the growth plate Linear bone growth results from chondrocyte proliferation and differentiation sequentially through the growth plate zones. FGFR3 is primarily expressed in proliferating chondrocytes, in which it tightly regulates proliferation and transition to terminal differentiation. Some FGFR3 is expressed in prehypertrophic chondrocytes, in which mutant FGFR3 dysregulates their differentiation and matrix secretion. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Achondroplasia: pathogenesis and implications Laederich and Horton 519 CNP is one of three structurally related peptides that were originally defined in the context of regulating blood pressure and volume . Through interaction with its receptor, natriuretic peptide receptor-B (NPR-B), CNP induces accumulation of intracellular cGMP. Signals downstream of NPR-B intersect with MAPK signals downstream of FGFR3 at the level of RAF1 (Fig. 1), antagonizing the growth-inhibiting effects of FGFR3 [48,49]. Both CNP and NPR-B are expressed in the proliferative and prehypertrophic zones of the growth plate, setting up a potential autocrine or paracrine regulatory circuit . Of interest, inactivating mutations of NPR-B are responsible for the dwarfing condition acromesomelic dysplasia, type Maroteaux (OMIM 602875) . Cells in many tissues express natriuretic peptide receptor-C (NPR-C) that lacks a functional intracellular domain and acts as ligand clearance receptor. In the growth plate, this decoy receptor appears to antagonize CNP–NPR-B signaling . Therapeutic targeting of Fibroblast Growth Factor Receptor 3 Many nonsurgical strategies aimed at reducing excessive FGFR3 output have been entertained as possible treatments to stimulate linear bone growth in achondroplasia. They include strategies to interfere with FGFR3 synthesis, block its activation, inhibit its tyrosine kinase activity, promote its degradation, and antagonize its downstream signals (Fig. 3). Many have been borrowed conceptually from the cancer treatment field, which is not surprising because the genetic lesions leading to achondroplasia and FGFR3-related skeletal disorders are identical to those found in FGFR3-driven cancers. Three of the proposed therapeutic strategies have progressed beyond the discussion phase and deserve more attention here: chemical inhibition of FGFR3 tyrosine kinase activity, antibody blockade of FGFR3 activation, and CNP-mediated antagonism of FGFR3 downstream signals. Fibroblast Growth Factor Receptor 3 kinase inhibitors The rationale behind this approach is that FGFR3’s kinase activity is critical for its activation and signal output; chemical inhibition of its kinase activity should block its inhibitory output and restore bone growth. The strategy has been successful for many oncogenic kinases . Aviezer et al.  reported synthesis of an inhibitor selective for FGFR3 compared with FGFR1. The compound reconstituted normal growth in cultured limb bones from a knock-in mouse model of achondroplasia. However, this success has not been extended to live mice. Figure 3 Potential targets for therapeutic intervention 1, FGFR3 tyrosine kinase; 2, ligand-mediated receptor activation; 3, CNP-mediated antagonism of signals downstream of receptor; 4, expression or synthesis of mutant FGFR3; 5, tyrosine kinase mediators of MAPK signaling pathway; 6, degradation of activated receptor. See text for discussion. Antibody blockade On the basis of the successful results from treating breast cancer patients with trastuzumab, this strategy was proposed for achondroplasia . Trastuzumab (trade name Herceptin) is a humanized monoclonal antibody to the extracellular domain of the HER2/neu (ErbB2), a transmembrane receptor overexpressed on the surface of about 15–30% of breast cancer cells . HER2 transmits mitogenic signals to the cancer cells in the absence of external mitogens, and trastuzumab reverses the adverse effects of the overactive receptor. Rauchenberger et al.  described generation of a comparable antibody to FGFR3 that effectively blocked ligand-induced FGFR3 activation; however, there have been no subsequent reports on its capacity to stimulate bone growth in vivo. Of note, activating FGFR3 mutations are relatively common in bladder cancer, in which they are thought to promote proliferation and survival of the tumor cells . Blocking antibodies similar to those just described have been used successfully to reduce proliferation in cells derived from these tumors, further supporting the utility of this approach to reducing FGFR3 output [57–59]. C-type natriuretic peptide antagonism of Fibroblast Growth Factor Receptor 3 signals The above-mentioned therapeutic strategy evolved from a series of mouse genetics investigations in the Nakao Laboratory in Kyoto, Japan dating back over a decade. The initial observation revealed that transgenic mice Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 520 Endocrinology and metabolism expressing high concentrations of brain natriuretic peptide (BNP) in the plasma exhibited postnatal skeletal overgrowth with elongation of long bone growth plates . Tibial organ culture experiments revealed that CNP was more potent than atrial natriuretic peptide (ANP) in stimulating growth, and that CNP mRNA was the only natriuretic peptide expressed in the tibias . Mice lacking BNP or ANP display no skeletal defects, whereas CNP knock-out mice exhibit severe postnatal dwarfism that was rescued when they were crossbred with mice overexpressing CNP from a transgene expressed only in cartilage . Together, these results suggest that CNP is the physiologic ligand for NPR-B in growing bones. These findings confirmed the ability of CNP to stimulate endochondral ossification in vivo. However, since the skeletal manifestations in the CNP null mouse resembled those of mouse models of achondroplasia, the question arose: can overexpression of CNP in cartilage rescue the dwarfism phenotype caused by achondroplasia mutations of FGFR3? To answer this question, transgenic mice overexpressing CNP in cartilage were crossed with mice displaying an achondroplastic phenotype resulting from overexpression of FGFR3 bearing the achondroplasia mutation in cartilage . The same Col2-a1 promoter was used to drive expression of both mutant FGFR3 and CNP. The growth deficiency of the achondroplastic mice was corrected by the local overexpression of CNP. A dose effect was demonstrated in that bone growth was greater in mice homozygous rather than heterozygous for the CNP transgene insertion, and the results suggested that the antagonism was primarily directed at MAPK-mediated FGFR3 signals. Additional studies have shown a dose response of the growth plate to circulating CNP produced in the liver from a liver-specific transgene promoter . Comparison of two different strains harboring the CNP transgene (one producing plasma concentrations approximately two-fold increased over normal, the other producing levels >2000-fold over normal of plasma CNP) revealed relatively greater growth response from the latter strain; however, the overgrowth led to deformities, such as severe kyphosis. Mice from this strain also exhibited reduced heart size consistent with cardiovascular consequences of such a high dose of CNP, but this complication was not detected in the mildly overexpressing transgenic mouse strain. The mildly overexpressing transgenic mice with a twofold sustained elevation of plasma CNP were analyzed further as a preclinical treatment model for achondroplasia . The results showed that the increased bone growth was associated with qualitatively normal skeletal radiographs and growth plate structure. Growth plate differentiation markers were essentially normal, although the zone thicknesses were slightly greater than wild type controls. These results are consistent with the notion that CNP ‘speeds up’ growth plate activity to generate more cartilage template over a given period of time, which translates into more bone growth. Further analysis of these mice also supported the postulate that CNP primarily modifies matrix production rather than chondrocyte proliferation or terminal differentiation. An additional observation was that these mice displayed a substantial increase in the growth of the foramen magnum size compared with controls. Most recently, the Nakao group has explored delivery of CNP through continuous intravenous infusion . These experiments bring to light the major obstacle in the potential therapeutic use of CNP to treat achondroplasia: its very rapid clearance from the plasma. The halflife of CNP in plasma is estimated to be 2.6 min . Rapid clearance was not a factor for the mice in which high CNP levels were sustained by overexpression of the respective transgenes in cartilage or liver; however, it becomes important if CNP is to be administered exogenously, as would most likely be the case for humans. The Nakao group administered recombinant CNP (CNP-22) as a continuous infusion into jugular veins of wild type and transgenic achondroplastic mice for 4 weeks starting at 3 weeks of age . Wild type mice treated at a dose of 1 mg/kg/min had 12% greater naso-anal lengths and more than 30% thicker growth plates than vehicleonly treated controls. Significant rescue of the bone growth deficiency was also observed after 3 weeks of treatment of the achondroplastic mice even at a 10-fold lower dose of CNP. Taken together, these investigations build a strong case for CNP as a viable therapeutic candidate for stimulating bone growth in achondroplasia. Systemic levels of CNP were achieved that promoted bone growth without causing obvious complications over a relatively short period of time in mice. CNP administration in humans has been described but only as transient infusions . More detailed analyses of side effects in mice over longer time frames will be needed, but the initial findings are promising. The greatest challenge will probably be extending the half-life and/or duration of effect of systemically administered CNP on bone growth . Therapeutic strategies to address this challenge might include chemical modifications of recombinant CNP to stabilize its structure or interfere with its normal clearance pathways such as antagonizing the decoy receptor NPR-C. Other possible treatment strategies Although not directed at mutant FGFR3, at least two other strategies have been explored to downregulate the consequences of similar activating mutations of FGFR2. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Achondroplasia: pathogenesis and implications Laederich and Horton 521 Apert syndrome, a classic and severe form of craniosynostosis, is commonly caused by the S252W mutation of FGFR2, which utilizes many of the same signaling pathways as FGFR3 to propagate its signals. Shukla et al.  utilized RNA interference to knock-down expression of the mutant FGFR2 allele. They generated a transgenic mouse strain that produced a short-hairpin RNA (shRNA) that reduced expression of transcripts from the mutant FGFR2 allele. When these mice were crossed with mice harboring a S252W FGFR2 allele and exhibiting features of Apert syndrome, the craniosynostosis phenotype was rescued. A similar approach targeted to the mutant FGFR3 allele in achondroplasia merits consideration, although delivery of an RNA interference vector to growth plate chondrocytes represents a challenge. Based on evidence that FGFR2 transmits signals through MAPK:MEK:ERK much as FGFR3 does, this group administered the MEK chemical inhibitor U0126 to the Apert syndrome mice prenatally and during the early postnatal period . The treatment partially ameliorated the craniosynostosis without obvious complications. These results raise the possibility of taking advantage of a similar strategy for achondroplasia, with the caution that MEK inhibition could affect many signaling pathways in addition to FGFR3 pathways . Growth plate targeting One potential approach to minimizing complications, while at the same time insuring local therapeutic levels of treatments, would be to target them to growth plate cartilage. This could be very important given the long duration that children with achondroplasia would presumably need to be treated, that is, infancy through puberty. This concern is relevant to all potential treatments for human growth plate disorders, including small molecule inhibitors and peptides such as CNP. Tissue targeting has not yet been developed for cartilage, but it is widely used in the pharmaceutical industry, mostly to target cancer [67,68]. In contrast to most cancers, growth plate cartilage is poorly vascularized. Nevertheless, it may be possible to target small therapeutic agents to growth plate chondrocytes by tagging them with small molecules that possess affinity for cartilage or chondrocytes. One such molecule, a short peptide, has been identified with affinity for cartilage matrix that might potentially be utilized for this purpose in the future . Another factor to consider is that the dense cartilage matrix that surrounds growth plate chondrocytes constitutes a diffusion barrier for systemically administered drugs . CNP and small molecule tyrosine kinase inhibitors are small enough that diffusion should not be an issue. However, it may limit the effectiveness of larger therapeutic agents such as monoclonal antibodies. Conclusions and future of achondroplasia treatment The molecular mechanisms underlying the pathology of achondroplasia make it a conceptually straightforward disorder to treat. Targeting a kinase has successfully been achieved for other diseases, such as cancer, and the pharmaceutical tools exist to target FGFR3. Despite this optimism, a decade of research has proven that a simplistic view of disease treatment is limited by the validity of our preclinical models, especially when potential treatments will be used long-term in children. Clinical trials for CNP and possibly the cancer-inspired therapeutics may well begin in the next 5 years; however, it may take years to determine their efficacy and safety. Hopefully, new advances into the targeting of therapies to the growth plate will reduce concerns over long-term side effects and accelerate translation of new therapies into the clinic. Acknowledgements The preparation of this review was supported by a grant from the Shriners Hospitals for Children (WAH). MBL is a student in the Graduate Program in Molecular and Cellular Biosciences at Oregon Health & Sciences University. She is a recipient of a Predoctoral Fellowship Award from the PhRMA Foundation. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 555). 1 Horton WA, Hall JG, Hecht JT. Achondroplasia. Lancet 2007; 370:162–172. 2 Velinov M, Slaugenhaupt SA, Stoilov I, et al. The gene for achondroplasia maps to the telomeric region of chromosome 4p. Nat Genet 1994; 6:314– 317. 3 Shiang R, Thompson LM, Zhu YZ, et al. Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell 1994; 78:335–342. 4 Bellus GA, Hefferon TW, Ortiz de Luna RI, et al. Achondroplasia is defined by recurrent G380R mutations of FGFR3. Am J Hum Genet 1995; 56:368–373. 5 Rousseau F, Bonaventure J, Legeai-Mallet L, et al. Mutations in the gene encoding fibroblast growth factor receptor-3 in achondroplasia. Nature 1994; 371:252–254. 6 Tavormina PL, Shiang R, Thompson LM, et al. Thanatophoric dysplasia (types I and II) caused by distinct mutations in fibroblast growth factor receptor 3. Nat Genet 1995; 9:321–328. 7 Bellus GA, McIntosh I, Smith EA, et al. A recurrent mutation in the tyrosine kinase domain of fibroblast growth factor receptor 3 causes hypochondroplasia. Nat Genet 1995; 10:357–359. 8 Colvin JS, Bohne BA, Harding GW, et al. Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat Genet 1996; 12:390– 397. 9 Deng C, Wynshaw-Boris A, Zhou F, et al. Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell 1996; 84:911–921. 10 Naski MC, Colvin JS, Coffin JD, Ornitz DM. Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3. Development 1998; 125:4977–4988. 11 Eswarakumar VP, Lax I, Schlessinger J. Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev 2005; 16:139–149. 12 Ornitz DM. FGF signaling in the developing endochondral skeleton. Cytokine Growth Factor Rev 2005; 16:205–213. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 522 Endocrinology and metabolism 13 Chen H, Ma J, Li W, et al. A molecular brake in the kinase hinge region regulates the activity of receptor tyrosine kinases. Mol Cell 2007; 27:717– 730. 38 Ben-Zvi T, Yayon A, Gertler A, Monsonego-Ornan E. Suppressors of cytokine signaling (SOCS) 1 and SOCS3 interact with and modulate fibroblast growth factor receptor signaling. J Cell Sci 2006; 119:380–387. 14 Itoh N, Ornitz DM. Functional evolutionary history of the mouse Fgf gene family. Dev Dyn 2008; 237:18–27. 39 Horton WA. Skeletal development: insights from targeting the mouse genome. Lancet 2003; 362:560–569. 15 Ornitz DM, Xu J, Colvin JS, et al. Receptor specificity of the fibroblast growth factor family. J Biol Chem 1996; 271:15292–15297. 40 Kronenberg HM. Developmental regulation of the growth plate. Nature 2003; 423:332–336. 16 Liu Z, Xu J, Colvin JS, Ornitz DM. Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18. Genes Dev 2002; 16:859–869. 41 Dailey L, Ambrosetti D, Mansukhani A, Basilico C. Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev 2005; 16:233–247. 17 Ohbayashi N, Shibayama M, Kurotaki Y, et al. FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis. Genes Dev 2002; 16:870–879. 18 Plotnikov AN, Schlessinger J, Hubbard SR, Mohammadi M. Structural basis for FGF receptor dimerization and activation. Cell 1999; 98:641–650. 19 Hart KC, Robertson SC, Donoghue DJ. Identification of tyrosine residues in constitutively activated fibroblast growth factor receptor 3 involved in mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation. Mol Biol Cell 2001; 12:931–942. 20 Mohammadi M, Olsen SK, Ibrahimi OA. Structural basis for fibroblast growth factor receptor activation. Cytokine Growth Factor Rev 2005; 16:107–137. 21 Horton WA, Degnin CR. FGFs in endochondral skeletal development. Trends Endocrinol Metab 2009; 20:341–348. 22 Su WC, Kitagawa M, Xue N, et al. Activation of Stat1 by mutant fibroblast growth-factor receptor in thanatophoric dysplasia type II dwarfism. Nature 1997; 386:288–292. 23 Legeai-Mallet L, Benoist-Lasselin C, Delezoide AL, et al. Fibroblast growth factor receptor 3 mutations promote apoptosis but do not alter chondrocyte proliferation in thanatophoric dysplasia. J Biol Chem 1998; 273:13007– 13014. 24 Sahni M, Ambrosetti DC, Mansukhani A, et al. FGF signaling inhibits chondrocyte proliferation and regulates bone development through the STAT-1 pathway. Genes Dev 1999; 13:1361–1366. 25 Hart KC, Robertson SC, Kanemitsu MY, et al. Transformation and Stat activation by derivatives of FGFR1, FGFR3, and FGFR4. Oncogene 2000; 19:3309–3320. 26 Choi DY, Toledo-Aral JJ, Lin HY, et al. Fibroblast growth factor receptor 3 induces gene expression primarily through Ras-independent signal transduction pathways. J Biol Chem 2001; 276:5116–5122. 27 Murakami S, Balmes G, McKinney S, et al. Constitutive activation of MEK1 in chondrocytes causes Stat1-independent achondroplasia-like dwarfism and rescues the Fgfr3-deficient mouse phenotype. Genes Dev 2004; 18:290– 305. 28 Zhang R, Murakami S, Coustry F, et al. Constitutive activation of MKK6 in chondrocytes of transgenic mice inhibits proliferation and delays endochondral bone formation. Proc Natl Acad Sci U S A 2006; 103:365–370. 29 Minina E, Kreschel C, Naski MC, et al. Interaction of FGF, Ihh/Pthlh, and BMP signaling integrates chondrocyte proliferation and hypertrophic differentiation. Dev Cell 2002; 3:439–449. 30 Minina E, Wenzel HM, Kreschel C, et al. BMP and Ihh/PTHrP signaling interact to coordinate chondrocyte proliferation and differentiation. Development 2001; 128:4523–4534. 31 Dailey L, Laplantine E, Priore R, Basilico C. A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation. J Cell Biol 2003; 161:1053–1066. 32 Horton WA. Skeletal Development. In: Unsicker K, Krieglstein K, editors. Cell signaling and growth factors in development. Weinheim: Wiley-VCH; 2006. pp. 619–640. 33 Webster MK, Donoghue DJ. Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia. EMBO J 1996; 15:520–527. 34 You M, Li E, Hristova K. The achondroplasia mutation does not alter the dimerization energetics of the fibroblast growth factor receptor 3 transmembrane domain. Biochemistry 2006; 45:5551–5556. 35 Monsonego-Ornan E, Adar R, Feferman T, et al. The transmembrane mutation G380R in fibroblast growth factor receptor 3 uncouples ligand-mediated receptor activation from down-regulation. Mol Cell Biol 2000; 20:516–522. 36 Cho JY, Guo C, Torello M, et al. Defective lysosomal targeting of activated fibroblast growth factor receptor 3 in achondroplasia. Proc Natl Acad Sci U S A 2004; 101:609–614. 37 Guo C, Degnin CR, Laederich MB, et al. Sprouty 2 disturbs FGFR3 degradation in thanatophoric dysplasia type II: a severe form of human achondroplasia. Cell Signal 2008; 20:1471–1477. 42 Murakami S, Kan M, McKeehan WL, de Crombrugghe B. Up-regulation of the chondrogenic Sox9 gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway. Proc Natl Acad Sci U S A 2000; 97:1113–1118. 43 Kratz CP, Zampino G, Kriek M, et al. Craniosynostosis in patients with Noonan syndrome caused by germline KRAS mutations. Am J Med Genet A 2009; 149A:1036–1040. 44 Tidyman WE, Rauen KA. The RASopathies: developmental syndromes of Ras/MAPK pathway dysregulation. Curr Opin Genet Dev 2009; 19:230– 236. 45 Yasoda A, Komatsu Y, Chusho H, et al. Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway. Nat Med 2004; 10:80–86. 46 Chusho H, Tamura N, Ogawa Y, et al. Dwarfism and early death in mice lacking C-type natriuretic peptide. Proc Natl Acad Sci U S A 2001; 98:4016– 4021. 47 Potter LR, Abbey-Hosch S, Dickey DM. Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions. Endocr Rev 2006; 27:47–72. 48 Miyazawa T, Ogawa Y, Chusho H, et al. Cyclic GMP-dependent protein kinase II plays a critical role in C-type natriuretic peptide-mediated endochondral ossification. Endocrinology 2002; 143:3604–3610. 49 Krejci P, Masri B, Fontaine V, et al. Interaction of fibroblast growth factor and C-natriuretic peptide signaling in regulation of chondrocyte proliferation and extracellular matrix homeostasis. J Cell Sci 2005; 118:5089–5100. 50 Yasoda A, Ogawa Y, Suda M, et al. Natriuretic peptide regulation of endochondral ossification. Evidence for possible roles of the C-type natriuretic peptide/guanylyl cyclase-B pathway. J Biol Chem 1998; 273:11695–11700. 51 Bartels CF, Bukulmez H, Padayatti P, et al. Mutations in the transmembrane natriuretic peptide receptor NPR-B impair skeletal growth and cause acromesomelic dysplasia, type Maroteaux. Am J Hum Genet 2004; 75:27–34. 52 Hartmann JT, Haap M, Kopp HG, Lipp HP. Tyrosine kinase inhibitors – a review on pharmacology, metabolism and side effects. Curr Drug Metab 2009; 10:470–481. 53 Aviezer D, Golembo M, Yayon A. Fibroblast growth factor receptor-3 as a therapeutic target for Achondroplasia – genetic short limbed dwarfism. Curr Drug Targets 2003; 4:353–365. 54 Hall PS, Cameron DA. Current perspective – trastuzumab. Eur J Cancer 2009; 45:12–18. 55 Rauchenberger R, Borges E, Thomassen-Wolf E, et al. Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3. J Biol Chem 2003; 278:38194–38205. 56 Cappellen D, De Oliveira C, Ricol D, et al. Frequent activating mutations of FGFR3 in human bladder and cervix carcinomas. Nat Genet 1999; 23:18– 20. 57 Martinez-Torrecuadrada J, Cifuentes G, Lopez-Serra P, et al. Targeting the extracellular domain of fibroblast growth factor receptor 3 with human singlechain Fv antibodies inhibits bladder carcinoma cell line proliferation. Clin Cancer Res 2005; 11:6280–6290. 58 Qing J, Du X, Chen Y, et al. Antibody-based targeting of FGFR3 in bladder carcinoma and t(4;14)-positive multiple myeloma in mice. J Clin Invest 2009; 119:1216–1229. 59 Hadari Y, Schlessinger J. FGFR3-targeted mAb therapy for bladder cancer and multiple myeloma. J Clin Invest 2009; 119:1077–1079. 60 Suda M, Ogawa Y, Tanaka K, et al. Skeletal overgrowth in transgenic mice that overexpress brain natriuretic peptide. Proc Natl Acad Sci U S A 1998; 95:2337–2342. 61 Kake T, Kitamura H, Adachi Y, et al. Chronically elevated plasma C-type natriuretic peptide level stimulates skeletal growth in transgenic mice. Am J Physiol Endocrinol Metab 2009; 297:E1339–E1348. This paper explores the physiologic consequences of sustained high levels of circulating CNP in mice. These effects are important to understand if CNP is to be used to treat humans. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Achondroplasia: pathogenesis and implications Laederich and Horton 523 62 Yasoda A, Kitamura H, Fujii T, et al. Systemic administration of C-type natriuretic peptide as a novel therapeutic strategy for skeletal dysplasias. Endocrinology 2009; 150:3138–3144. This article presents results of systemic administration of CNP in a mouse model of achondroplasia. 63 Hunt PJ, Richards AM, Espiner EA, et al. Bioactivity and metabolism of C-type natriuretic peptide in normal man. J Clin Endocrinol Metab 1994; 78:1428– 1435. 64 Lee CY, Chen HH, Lisy O, et al. Pharmacodynamics of a novel designer natriuretic peptide, CD-NP, in a first-in-human clinical trial in healthy subjects. J Clin Pharmacol 2009; 49:668–673. This report describes the first trial of CNP in humans. The genetically engineered CNP was tailored for therapeutic effects on heart failure in adults and infusions were for only 4 h. Nevertheless, the report reflects the initial use of CNP as a therapeutic agent in humans. 65 Shukla V, Coumoul X, Wang RH, et al. RNA interference and inhibition of MEK–ERK signaling prevent abnormal skeletal phenotypes in a mouse model of craniosynostosis. Nat Genet 2007; 39:1145–1150. 66 Yoon S, Seger R. The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 2006; 24: 21–44. 67 Goldberg M, Langer R, Jia X. Nanostructured materials for applications in drug delivery and tissue engineering. J Biomater Sci Polym Ed 2007; 18:241–268. 68 Alexis F, Pridgen E, Molnar LK, Farokhzad OC. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm 2008; 5:505– 515. 69 Rothenfluh DA, Bermudez H, O’Neil CP, Hubbell JA. Biofunctional polymer nanoparticles for intra-articular targeting and retention in cartilage. Nat Mater 2008; 7:248–254. 70 Morris NP, Keene DR, Horton WA. Morphology and chemical composition of connective tissue: cartilage. In: Royce PM, Steinmann B, editors. Connective tissue and its heritable disorders. 2nd ed. New York: Wiley; 2002. pp. 41–66. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.