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Transcript
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 [1]. 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
[14]. 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 [35] 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. [38] 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 [47]. 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 [50]. Of interest, inactivating mutations of
NPR-B are responsible for the dwarfing condition acromesomelic dysplasia, type Maroteaux (OMIM 602875)
[51].
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 [47].
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
[52]. Aviezer et al. [53] 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 [53]. 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 [54]. 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.
[55] 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 [56].
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
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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
[60]. 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 [50]. 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 [46]. 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 [45]. 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 [61]. 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 [61]. 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 [62].
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 [63].
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 [62]. 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 [64]. 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 [1]. 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. [65] 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 [65]. 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 [66].
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 [69].
Another factor to consider is that the dense cartilage
matrix that surrounds growth plate chondrocytes constitutes a diffusion barrier for systemically administered
drugs [70]. 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.
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Achondroplasia: pathogenesis and implications Laederich and Horton 523
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