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Journal of Oral Biosciences 59 (2017) 121–126
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Review
Molecular mechanisms for activation of mutant activin receptor-like
kinase 2 in fibrodysplasia ossificans progressiva
Mai Fujimoto a,b,n, Naoto Suda b, Takenobu Katagiri a,nn
a Division of Pathophysiology, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka-shi, Saitama 350-1241, Japan
b Division of Orthodontics, Department of Human Development and Fostering, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado-shi,
Saitama 350-0283, Japan
a r t i c l e i n f o
Article history:
Received 3 February 2017
Received in revised form
7 March 2017
Accepted 11 March 2017
Available online 13 April 2017
Keywords:
Fibrodysplasia ossificans progressiva
BMP
ALK2
Receptors
x.doi.org/10.1016/j.job.2017.03.004
79/& 2017 Japanese Association for Oral Biolo
reativecommons.org/licenses/by-nc-nd/4.0/).
esponding author at: Division of Orthodontic
ent and Fostering, Meikai University School
shi, Saitama 350-0283, Japan.
responding author at: Division of Pathophy
c Medicine, Saitama Medical University, 1397
0‐1241, Japan.
ail addresses: m.fujimoto@dent.meikai.ac.jp (M
@saitama-med.ac.jp (T. Katagiri).
a b s t r a c t
Background: Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder char-
acterized by progressive heterotopic ossification (HO) in soft tissues such as skeletal muscles, tendons,
and ligaments. Gain-of-function mutations in ALK2, a type I receptor for bone morphogenetic proteins
(BMPs), have been identified in patients with FOP; however, the molecular mechanisms that result in the
activation of ALK2 mutants remain unclear.
Highlight: FOP-associated mutant ALK2 signaling is further enhanced by BMP type II receptor in a serine/
threonine kinase activity-dependent manner. The threonine residue at position 203 (T203) in ALK2 is
crucial for this enhancement by BMP type II receptor. Recently, activin A was found to be a critical ligand
of ALK2 mutants.
Conclusion: BMP type II receptor-dependent phosphorylation of ALK2 mutants in response to ligand
binding is important for the activation of BMP signaling in FOP. Therefore, the use of anti-activin A
compounds would be a novel treatment approach for FOP.
& 2017 Japanese Association for Oral Biology. Published by Elsevier B.V. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Bone tissues are formed in vertebrates via two related but distinct
pathways: intramembranous ossification and endochondral ossifica-
tion. In intramembranous ossification, undifferentiated mesenchymal
cells differentiate into osteoblasts, which are unique bone-forming
cells. In contrast, in endochondral ossification, chondrocytes develop
from undifferentiated mesenchymal cells, followed by the differ-
entiation of osteoblasts on the cartilaginous templates.
Pathological heterotopic ossification (HO) can be induced in
soft tissues, such as skeletal muscles and subcutaneous tissues, by
implantation of bone morphogenetic protein (BMP) in vivo. Fi-
brodysplasia ossificans progressiva (FOP; MIM135100) is a rare
autosomal dominant hereditary disorder characterized by post-
natal progressive HO through endochondral ossification in soft
tissues such as skeletal muscles, tendons, and ligaments (Table 1)
gy. Published by Elsevier B.V. This
s, Department of Human De-
of Dentistry, 1-1 Keyakidai,
siology, Research Center for
-1 Yamane, Hidaka-shi, Sai-
. Fujimoto),
[1–4]. Although the gene responsible for FOP was identified in
2006, there is still no effective treatment approved for preventing
HO in FOP [5]. Therefore, the elucidation of the molecular me-
chanisms underlying HO in FOP would contribute to the devel-
opment of a novel treatment method for FOP.
2. FOP
The clinical features observed in patients with FOP are sum-
marized in Table 1. The incidence of FOP is estimated at 1 in
2 million people regardless of race, gender, age, or living condition
in various countries. Patients with typical FOP have congenital
malformation of the great toes at birth and show HO in early
childhood [1–4]. In a study by Nakashima et al. [6], more than 93%
of the feet (29 out of 31) of Japanese patients with FOP showed
great toe deformity. Biopsies and surgical treatments are pro-
hibited for FOP because HO is induced by muscle injury including
accidental trauma [1–4]. Viral infection is also known to induce HO
in patients with FOP [1–4]. HO induces the fusion of tempor-
omandibular joints and a reduced range of joint motion [7]. Al-
though HO occurs in skeletal muscles, it is not observed in the
tongue, eyelid muscles, or diaphragm. In patients with FOP, a
discordant intermaxillary relationship is caused by hypoplasia of
the mandibular condyle and mandible [7]. Moreover, such cases of
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Table 1
Clinical features of FOP.
� Progressive HO in soft tissues (skeletal muscles, tendons, and ligaments)
� Congenital malformation of great toes at birth
� Muscle trauma and viral infection induce acute HO
� Autosomal dominant disorder
� Affects 1 in 2 million people
� Mutations in ALK2/ACVR1
M. Fujimoto et al. / Journal of Oral Biosciences 59 (2017) 121–126122
HO and joint fusion restrict mouth opening, resulting in poor oral
hygiene and leading to caries, gingivitis, and periodontitis [8,9].
3. BMPs and their intracellular signaling
In the early 1990s, BMP signaling was suggested to be involved
in FOP because BMPs are unique growth factors that can induce
HO in soft tissues such as skeletal muscles [10]. BMPs are members
of the transforming growth factor-β (TGF-β) family and are im-
portant growth factors for bone formation in various vertebrates
[11]. The TGF-β family contains more than 30 members including
BMPs, activins, growth and differentiation factors (GDFs), and the
TGF-βs themselves [11–13]. TGF-β family members have been
examined for HO-inducing activity in vivo by implantation of
purified or recombinant proteins into skeletal muscles in verte-
brates. Although several BMPs, including BMP-2, BMP-4, BMP-6,
BMP-7, and BMP-9, induced HO in vivo, TGF-β1, activin, and BMP-3
failed to induce HO [11–14]. The osteogenic and non-osteogenic
activities of members of the TGF-β family have also been de-
monstrated in vitro using C2C12 myoblasts [14]. In C2C12 cells,
most of the osteogenic and non-osteogenic ligands inhibited
myogenic maturation; however, only osteogenic ligands induced
osteoblastic differentiation [15]. These findings suggest that an
Fig. 1. Signal transduction by TGF-β family members. When a ligand binds to both type
kinase phosphorylates the GS domain of type I receptor as a substrate to activate th
transcription factors such as Smad proteins. The phosphorylated Smad proteins transloca
target gene.
osteogenic ligand of the TGF-β family or a downstream signaling
molecule may be mutated and over-activated in patients with FOP.
The signaling system is conserved among osteogenic and non-
osteogenic members of the TGF-β family (Fig. 1). First, ligands bind to
two types of transmembrane serine (Ser)/threonine (Thr) kinase re-
ceptors located on the cell membrane of target cells (Fig. 1) [11–14].
TypeII receptors (BMPR-II, ActR-IIA, ActR-IIB, and TβR-II) are con-
stitutively activated kinases regardless of ligand binding and phos-
phorylate type I receptors as substrates [11–14]. In contrast to type II
receptors, type I receptors (ALK1 through ALK7) are inactive kinases;
their enzymatic activity is activated through phosphorylation by type
II receptor kinases (Fig. 1) [11–14]. The activated type I receptors
phosphorylate Smad proteins [Smad1, Smad2, Smad3, Smad5, and
Smad9 (also known as Smad8)], which then translocate to the nu-
cleus and bind to specific sequences in the enhancer regions of target
genes to regulate their transcription (Fig. 1) [11–14].
Osteogenic ligands, such as BMPs and GDFs, bind to ALK1, ALK2,
ALK3, or ALK6 and induce the phosphorylation of Smad1, Smad5,
and Smad9, whereas non-osteogenic ligands, such as TGF-βs and
activins, bind to ALK4, ALK5, or ALK7 and activate Smad2 and
Smad3 (Fig. 2) [11–14]. Type II receptors, particularly ActR-IIA and
ActR-IIB, can interact with both osteogenic and non-osteogenic
ligands, suggesting that the biological activities of ligands are
determined by type I receptors rather than type II receptors (Fig. 2)
[11–14]. Activin A, a non-osteogenic ligand, was found in previous
studies to bind to ALK2 in vitro; however, the physiological role of
this binding remains unclear as intracellular signaling seems to be
transduced mainly via ALK4 but not ALK2 [16,17].
4. Association of ALK2 mutations with genetic disorders
Most patients with sporadic and familial FOP have a hetero-
zygous "G" to "A" substitution in the ACVR1 gene at position 617
I and type II receptors located on the cell membrane of target cells, type II receptor
e kinase activity of type I receptor. Then, type I receptor kinase phosphorylates
te to the nuclei and bind to a specific DNA sequence to regulate the transcription of a
Fig. 2. Osteogenic and non-osteogenic members of the TGF-β family signaling molecules. The molecular mechanisms of intracellular signal transduction for both osteogenic
and non-osteogenic ligands are conserved among the TGF-β family. Some type II receptors, such as ActR-IIA and ActR-IIB, interact with the same transduction molecules. In
contrast, the downstream molecules of type I receptors are highly specific, suggesting that type I receptors determine the biological activity and specificity of the ligands.
M. Fujimoto et al. / Journal of Oral Biosciences 59 (2017) 121–126 123
(c.617G4A) [5]. The ACVR1 gene encodes ALK2, and the c.617G4A
mutation causes the substitution of an arginine residue at position
206 with a histidine residue (p.R206H) in ALK2 (Fig. 3) [5].
In addition to p.R206H, more than 10 other ALK2 mutations
have been identified in patients with FOP who have different
clinical features (atypical FOP) (Fig. 3) [18]. All of the mutations
associated with FOP can be mapped to the intracellular domain of
ALK2 (Fig. 3). The mutations p.R206H, p.L196P, p.
Fig. 3. Mutations of ALK2 in patients with fibrodysplasia ossificans progressiva (FOP),
mutations identified in patients with FOP, DIPG, and HD are indicated by black circles. M
and kinase domain) except for A15G, which is associated with HD.
P197_F198delinsL, p.R202I, and p.Q207E are localized in the "GS
domain" of ALK2, which is the phosphorylation site for BMP type II
receptor kinases. Other mutations, such as p.R258G/S, p.G325A, p.
G328E/R/W, p.G356D, and p.R375P, are localized in the serine/
threonine kinase domain of ALK2 (Fig. 3). In addition, the ALK2
mutations p.A15G, p.H286N, p.R307L, and p.L343P have been
found in patients with severe heart defects (Fig. 3) [19,20]. Somatic
mutations of ALK2, including p.R206H, p.Q207E, p.R258S/G, p.
diffuse intrinsic pontine glioma (DIPG), and heart defects (HD). The types of ALK2
ost of the mutations are located in the intracellular domain of ALK2 (the GS domain
M. Fujimoto et al. / Journal of Oral Biosciences 59 (2017) 121–126124
G328E/W, and p.G356D, have been found in patients with diffuse
intrinsic pontine glioma (DIPG), a type of brain tumor (Fig. 3) [21–
24]. A novel mutation, p.G328V, was identified in a patient with
DIPG but not in patients with FOP or heart defects (Fig. 3) [21–24].
All of the ALK2 mutations associated with FOP and DIPG (ex-
cluding heart defects) could induce Smad1/5-dependent BMP in-
tracellular signaling in vitro without the addition of exogenous
BMP, suggesting that the gain-of-function mutations of ALK2 are
involved in both FOP and DIPG (Fig. 3). In contrast, the heart de-
fect-associated ALK2 mutants could not induce BMP signaling
even with type II receptor co-expression, suggesting that they are
loss-of-function mutations (Fig. 3).
Variations in ALK2 mutations appear to determine the clinical
features of patients with FOP. For example, in comparison with
patients with typical FOP carrying the p.R206H mutation, a patient
with p.L196P did not have either great toe malformations at birth
or HO in skeletal muscles until a motorcycle accident at the age of
21 [25]. Another patient who had a novel c.974G4C (p.G325A)
mutation in ALK2 did not have HO (even after muscle injury) until
a viral illness at the age of 47 [26].
Fig. 4. Effect of Ser and Thr substitution mutations in the ALK2 GS domain on type
II receptor activation. The amino acid sequence of the GS domain of human ALK2
(from S178 to T209) is shown. The GS domain contains five Ser residues and four
Thr residues as potential phosphorylation sites of type II receptor Ser/Thr kinases.
In the 9AV mutant, all Ser and Thr residues are substituted with Ala and Val re-
sidues, respectively. In the 8AV mutant, eight Ser and Thr residues (except T203)
are substituted with Ala and Thr residues, respectively. In the T203V mutant, T203
is substituted with a Val residue. The activation of each type of ALK2 by type II
receptors is indicated by þ or - in the figure.
5. Enhancement of FOP-associated mutant ALK2 signaling by
BMP type II receptor kinases
Although patients with FOP have germline mutations in ALK2,
they do not have HO at birth; HO is only induced following muscle
trauma and/or viral infection. These clinical features of FOP sug-
gest that a molecular mechanism in addition to the genetic mu-
tation of ALK2 is involved in HO induction. Indeed, the BMP ac-
tivity induced by overexpression of mutant ALK2 in vitro has been
reported to be weak, thus suggesting that an additional mechan-
ism enhances mutant ALK2 signaling.
Since BMP type II receptors is a direct activator of type I re-
ceptors including ALK2, the effect of this receptor on the BMP
signaling induced by FOP-associated ALK2 mutants has been in-
vestigated. The biological activity of mutant ALK2 identified in
patients with FOP was enhanced by co-expression with BMPR-II or
ActR-IIB but not with ActR-IIA or TβR-II [27]. Similar results were
reported in Drosophila [28]. Neither BMPR-II nor ActR-IIB was
found to activate any of the ALK2 mutants associated with heart
disease because they are loss-of-function mutations [27]. However,
in contrast to the wild-type, the kinase-dead (KR) mutants of
BMPR-II or ActR-IIB did not enhance BMP signaling by FOP-asso-
ciated ALK2 mutants, suggesting that the enhanced signaling by
type II receptors is a kinase activity-dependent event [27]. More-
over, the signaling of the p.R206H mutant was enhanced by both
BMPR-II and ActR-IIB, whereas that of the p.G325A mutant was
enhanced by ActR-IIB only and not by BMPR-II [27]. Because the p.
G325A mutation was identified in a patient with late-onset FOP,
the sensitivity of ALK2 mutants to BMP type II receptors seems to
determine the clinical features of FOP, especially HO [27].
The enhancement of ALK2 mutants by type II receptors is highly
dependent on the Ser/Thr kinase activity of type II receptors, sug-
gesting that ALK2 mutants are phosphorylated as substrates. The GS
domain of type I receptors is the site phosphorylated by type II
receptors of the TGF-β family [11–14]. Human ALK2 has five Ser and
four Thr residues in the GS domain(Fig. 4). Signaling enhancement
by type II receptors has been reported to be abolished in a mutant
in which all nine Ser and Thr residues were substituted with Ala
and Val, respectively, thus suggesting that the GS domain is the
phosphorylation site for type II receptor kinases (Fig. 4) [27]. Fur-
thermore, a series of substitution mutations in the ALK2 GS domain
identified the crucial role of the Thr residue at position 203 (T203)
in type II receptor-dependent activation of ALK2. The 8AV mutation,
in which eight out of nine Ser/Thr residues (except T203) were
mutated to Ala/Val residues, induced BMP activity in the presence
of type II receptors (Fig. 4) [27]. In contrast, a single Thr to Val re-
sidue substitution at position 203 (T203V) abolished BMP signaling
even in the presence of type II receptors (Fig. 4) [27]. The Thr re-
sidue at position 203 in ALK2 is conserved in various species in-
cluding H. sapiens, M. musculus, R. norvegicus, C. familiaris, B. taurus,
G. gallus, X. laevis, D. rerio, and F. rubripes [27]. Moreover, this Thr
residue is conserved among all BMP type I receptors, such as in
ALK1, ALK3, and ALK6 at positions 197, 229, and 199, respectively,
and it is essential for the activation of intracellular signal trans-
duction in response to ligand stimulation [27]. Considering that the
ALK2 phosphorylation of the T203V mutant was found to be lower
than that of wild-type T203 in the presence of type II receptors,
T203 is suggested to regulate the phosphorylation levels of ALK2
induced by type II receptors [27].
Phosphorylation of the GS domain by type II receptors may
induce changes in the three-dimensional structure, as may inter-
action with the activators and inhibitors of the kinase. FKBP12 is a
binding protein of the immunosuppressive drug FK506 and binds
to the unphosphorylated GS domains of type I receptors [29]. The
interaction between FKBP12 and type I receptors can be abolished
in response to ligand stimulation in a type II receptor kinase-de-
pendent manner [29,30]. FKBP12 has been suggested to be in-
volved in the onset of HO in patients with FOP because the in-
teraction with FKBP12 was reduced when ALK2 was mutated [31].
It is possible that type II receptors phosphorylate the GS domain,
abolish the interaction with FKBP12, and enhance intracellular
signaling even when ALK2 is mutated in FOP. Further studies are
needed to elucidate the role of FKBP12 in FOP.
6. Development of potential treatments for FOP
The identification of gain-of-function mutations in ALK2 re-
sponsible for FOP indicated that ALK2 inhibitors would be poten-
tial treatment options for FOP [32]. Because ALK2 is a Ser/Thr ki-
nase, various types of small-molecule kinase inhibitors have been
developed to block the intracellular signaling induced by ALK2
mutants [33,34]. An allele-specific RNA interference technique was
established to specifically inhibit mutated ALK2 in FOP [35]. In
addition, small-molecule inhibitors of osteoblastic differentiation
induced by BMP signaling were identified in a screen using an
ALK2 mutant, p.R206H [36].
Recently, activin A, which does not show HO-inducing activity
in vivo, was found to be involved in HO in FOP. The p.R206H
mutant but not wild-type ALK2 phosphorylated Smad1 and Smad5
and induced the transcription of their target gene in response to
activin A stimulation [37]. In an inducible knock-in mouse model
of p.R206H, HO was induced within 2–4 weeks after the induction
Fig. 5. Induction of BMP signaling in FOP by type II receptor kinase phosphoryla-
tion of mutant ALK2. Although FOP-associated ALK2 mutants have gain-of-function
mutations, they still require the phosphorylation of the GS domain by type II re-
ceptors in response to ligand stimulation such as by activin A. The T203 residue
plays an important role in regulating the phosphorylation of ALK2 by type II re-
ceptor kinases.
M. Fujimoto et al. / Journal of Oral Biosciences 59 (2017) 121–126 125
of mutant ALK2 expression. In this mouse model, HO was inhibited
by dominant-negative type II receptors or anti-activin A antibodies
[37]. In addition, mesenchymal stromal cells prepared from in-
ducible pluripotent stem cells (iPSCs), which were established
from patients with FOP, formed HO when they were transplanted
with activin A-expressing cells in immunodeficient mice [38].
These findings suggest that activin A is a trigger of HO in FOP.
Therefore, anti-activin A compounds acting in the extracellular
space are potential new candidates for the development of ther-
apeutic drugs for FOP.
7. Conclusions
In 2006, ALK2 was identified as the molecule responsible for
FOP. More than 10 mutations in ALK2 have been found in patients
with typical and atypical FOP. Initially, mutant ALK2 was thought
to be constitutively activated by BMP type I receptors because of
gain-of-function mutations. However, it is a mildly activated mu-
tant and can be further activated in response to ligand stimulation,
suggesting that an additional molecular mechanism is involved in
the HO in FOP. This possibility is further supported by the clinical
features of FOP, which show that HO is inducible after muscle
trauma or viral infection even with germline genetic mutations.
Signaling by the various FOP-associated ALK2 mutants can be
further enhanced by type II receptors in a phosphorylation-de-
pendent manner. The phosphorylation levels of ALK2 are regulated
by T203 in ALK2. Moreover, the identification of activin A as a
critical ligand in FOP also supports the importance of type II re-
ceptor-dependent phosphorylation of mutant ALK2 for BMP sig-
naling activation in FOP (Fig. 5). These findings will contribute to
the development of novel treatment methods for FOP in the
future.
Ethical approval
Ethical approval is not required for this review.
Conflict of interest
MF and NS declare no conflict of interest. TK received a research
grant from Daiichi-Sankyo Co., Ltd.
Acknowledgments
We thank the members of the Division of Pathophysiology,
Research Center for Genomic Medicine, Saitama Medical Uni-
versity for their valuable discussions. This work was supported in
part by a grant-in-aid from the Support Project for the Formation
of a Strategic Center in a Private University from the Ministry of
Education, Culture, Sports, Science and Technology of Japan [JSPS
KAKENHI No. 25293326 and 17H04317 (TK) and 25293422 (NS)]
and a Miyata Research Grant from Meikai University (MF).
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	Molecular mechanisms for activation of mutant activin receptor-like kinase 2 in fibrodysplasia ossificans progressiva
	Introduction
	FOP
	BMPs and their intracellular signaling
	Association of ALK2 mutations with genetic disorders
	Enhancement of FOP-associated mutant ALK2 signaling by BMP type II receptor kinases
	Development of potential treatments for FOP
	Conclusions
	Ethical approval
	Conflict of interest
	Acknowledgments
	References