The BMPs comprise a group of more than 15 ligands, and is the largest TGF‐β subfamily. Based on sequence simi- larities and their diversity of functions, this subfamily can be further subdivided into BMP‐2/‐4, BMP‐5/‐6/‐7/‐8, BMP‐9/‐10, and BMP‐12/‐13/‐14 (growth and differentia- tion factor, GDF‐7/‐6/‐5, respectively) subgroups. The roles of BMP pathway components in bone formation have been summarized in a recent review [12]. The key findings are summarized here.
BMP‐2
An osteoblast‐specific disruption of Bmp2 using 3.6 kilobase (kb) Col1a1‐Cre leads to thinner bones in mice with increased brittleness. Loss of Bmp2 in limb mesenchyme leads to a greatly increased rate of fracture owing to defects in the periosteum and ability to mount a reparative response ([12] and citations therein). Chondrocyte‐specific disrup- tion of Bmp2 or both Bmp2 and Bmp4 leads to severe defects in chondrocyte proliferation and maturation during
BMPS and Bone Development 63 endochondral bone formation, while chondrocyte‐specific
disruption of only Bmp4 causes minor changes in chondro- cyte maturation [13]. This indicates that BMP‐2 has a cru- cial and nonredundant role in chondrocyte proliferation and maturation during endochondral bone development, as well as in fracture repair.
BMP‐4
Limb‐mesenchyme‐specific knockouts of Bmp2 and Bmp4 demonstrate that these ligands function redun- dantly to control osteogenesis in the limb [14]. Osteoblastic differentiation and osteogenesis are defective in Bmp2 and Bmpr4 conditional (Prx1‐Cre) knockout mice, but these defects can be rescued by one functional allele of either Bmp2 or Bmp4 ([12] and citations therein). Consistent with an anabolic role for BMPs in promotion of bone remodeling, overexpression of Bmp4 in osteoblasts results in an increase of osteoclastogenesis and a reduction in bone mass [15].
BMP‐5/‐6/‐7
Mutation in Bmp5 is associated with a wide range of skeletal defects, including reductions in long bone width, the size of several vertebral processes, and an overall lower body mass [16].
BMP‐6 is highly expressed in hypertrophic chondro- cytes. However, Bmp6–/– mice show only a slight delay in the ossification of the sternum and a reduction in the size of long bones, which is slightly exacerbated in Bmp5/Bmp6 double mutants [17]. Compound knockout mice lacking one allele of Bmp2 and both alleles of Bmp6 (Bmp2+/–
;Bmp6–/–) exhibit moderate growth retardation, showing a reduction in trabecular bone volume with suppressed bone formation, while the single deficient mice (Bmp2+/–
or Bmp6–/–) do not [18]. Thus, there is a considerable func- tional overlap at the level of BMP ligands, and the combination of BMP‐2 and BMP‐6 plays a pivotal role in bone formation.
Bmp7 homozygous null mice exhibit an early postna- tal lethality mutation that is associated with various developmental defects: holes in the basisphenoid bone and xiphoid cartilage, retarded ossification of bones, fused ribs and vertebrae, underdeveloped neural arches of the lumbar and sacral vertebrae, and polydactyly of the hindlimbs [19]. On the other hand, conditional deletion of Bmp7 from the limb has only minor consequences ([12] and citations therein). These divergent findings sug- gest that BMP‐7 may have redundant roles with other BMPs in bone formation and may have a more prominent role in axial than in appendicular elements.
BMP‐13/GDF‐6 and BMP‐14/GDF‐5/CDMP‐1
Mutations in the BMP‐13, ‐14, and ‐15 subgroups are associated with fusions of joints and defects in cranial suture formation that are not seen in mice lacking other
BMP family members. Mutation in Bmp13 causes defects at multiple sites, including joint fusions in the wrist and ankle, fusions of carpal and tarsal bones, cartilage defects in the middle ear, and the absence of the coronal suture [20]. Bmp13 knockout mice have accelerated coronal suture fusion, indicating an inhibitory role of BMP‐13 in osteogenic differentiation [21]. Bmp13 knockout mice also exhibit shorter dermal flat bones in the skull and shorter digits [22]. It is also shown that a hindlimb enhancer in the Bmp13 locus has a striking correlation with known changes in hindlimb digit length and mus- culature that have evolved during the transition to bipedal locomotion in the human lineage [22]. The joints altered in Bmp13–/– mice are distinct from those altered in Bmp14 mutants, and Bmp13/Gdf6;Bmp14/Gdf5 dou- ble mutants show additional defects.
BMP‐14/GDF‐5 has a fundamental role in limb devel- opment, where it controls the size of the initial cartilagi- nous condensations as well as the coordination of bone and joint formation. Mutations of Bmp14/Gdf5 in mice cause brachypodism, reduction of digit number, fusion of some bones in the wrist and ankle, ankylosis of the knee joint and malformation with early onset osteoarthritis of the elbow joint [23]. Transgenic mice expressing Bmp14/Gdf5 under the control of a Col11a2 promoter show extensive cartilage overgrowth and complete absence of joints [24]. How members of this subgroup promote joint formation on the one hand, but promote chondrogenesis on the other, is not understood.
TG/KO phenotypes of receptors
BMP ligands transduce their signals through complexes composed of type I and type II serine/threonine kinase recep- tors. BMPs bind to three distinct type I receptors, called activin receptor‐like kinase 2 (ALK‐2, also known as activin receptor type I, ACVR1), ALK‐3 (also known as BMP recep- tor type IA, BMPR1A), and ALK‐6 (also known as BMP receptor type IB, BMPR1B). BMP receptor type II (BMPRII), activin receptor type II (ACVRII), and activin receptor type IIB (ACVRIIB) serve as type II receptors for BMPs.
ACVR1/ALK‐2
Deletion of Acvr1 using a 3.2‐kb Col1‐CreER increases bone mass in association with suppression of Sost and Dkk1 [25]. Mice deficient for Acvr1 in chondrocytes, achieved using a Col2‐Cre driver, exhibit a shortened cra- nial base and hypoplastic cervical vertebrae [26].
Activation experiments showed that BMPR1A, BMPR1B, and ACVR1 are all able to promote chondrogenesis.
However, deletion of each individual receptor only results in mild skeletal defects or defects restricted to isolated skeletal elements [26,27]. Bmpr1a/Bmpr1b double mutant mice, Acvr1/Bmpr1a double mutant mice, and Acvr1/Bmpr1b double mutant mice exhibit generalized chondrodysplasia that is much more severe than any of
the corresponding mutant strains [26,27]. Unlike com- pound mutant mice for Bmpr1a and Bmpr1b, compound mutant mice for Avcr1 and Bmpr1b can develop cartilage primordia and subsequent bones through endochondral ossification [26], suggesting that BMP signaling through ACVR1 plays a relatively minor role compared with other type I receptors during chondrogenesis.
BMPR1A/ALK‐3
Deletion of Bmpr1a using a 3.2‐kb Col1‐CreER increases bone mass due to decreased osteoblast activity and more dramatically decreased osteoclast activity [28]. The con- stitutively active form of Bmpr1a (caBmpr1a) is associ- ated with a partial rescue of the bone phenotype of Bmpr1a‐deficient mice [28]. Conditional disruption of Bmpr1a using Dmp1‐Cre demonstrates an increased bone mass concomitant with accelerated cell proliferation and Sost reduction [29,30]. When Bmpr1a is conditionally disrupted in osteoclasts using a Cathepsin K (CtsK) pro- moter, bone mass increases [31].
Disruption of Bmpr1a in chondrocytes demonstrates impairment of articular cartilage and growth plate cartilage, resulting in decreased bone size and bone mass [27]. The loss of both BMPR1A and BMPR1B blocks chondrocyte condensation, proliferation, differentiation, survival, and function due to impaired Sox expression [27]. These mouse models demonstrate that BMP signaling is essential for almost every step during endochondral bone development.
The constitutively active form of Bmpr1a (caBmpr1a) in neural crest cells results in craniosynostosis in mice [28,32], as well as bone and cartilage defects of the naso- maxillary complex, due to an increased level of cell death in skeletal primordia [32,33].
BMPR1B/ALK‐6
Unlike in ACVR1 and BMPR1A mice, mice homozygous null for Bmpr1b are viable. In Bmpr1b‐deficient mice, proliferation of prechondrogenic cells and differentiation of chondrocytes are markedly reduced, leading to reduced lengths in the phalangeal region [34]. In addition, bone mass in the mutant mice is decreased, in association with compromised osteoblastic differentiation of bone marrow mesenchymal progenitors [35].
In Bmpr1b and Bmp7 double mutant mice, severe skel- etal defects are observed in the forelimbs and hindlimbs [34]. Since BMP‐7 binds efficiently to both BMPR1B and ACVR1, it is conceivable that BMPR1B and ACVR1 play important synergistic or overlapping roles in cartilage and bone formation in vivo. Conditional disruption of Bmpr1a driven by Col2‐Cre and Bmpr1b homozygous null double mutant mice exhibit a dramatic decrease in the size of skeletal primordia (i.e. chondrodysplasia) due to a reduction of proliferation and an increase in apopto- sis around E12.5 to E16.5 [27]. This suggests a possible functional compensation mechanism between BMPR1A
and BMPR1B in chondrocytes during early cartilage development in growth plates [27].
BMPRII
Deletion of Bmpr2 using Prx1‐Cre results in normal bone development both at embryonic stages and at birth, prob- ably due to compensation by the other type II receptors, ACVRII and ACVRIIB, suggesting that BMPRII is not required for endochondral ossification in the limb.
However, the mutant mice have increased bone mass at 2 months after birth [36]. In this mouse model, BMP sign- aling is unchanged, whereas activin signaling is impaired, leading to increased osteoblast activity. Activins (which bind to type I activin receptors and lead to phosphoryla- tion of Smad2 and ‐3) and BMPs (which bind to ACVR1, BMPR1A, and BMPR1B and lead to phosphorylation of Smad1 and ‐5) can all transduce their signals through receptor complexes that contain ACVRII and ACVRIIB.
This study therefore suggests that type II receptor segre- gation and/or competition could be a generalized mecha- nism by which BMP and activin signaling interact.
Disease connection
Given the important roles of BMP signaling in chondro- genesis and osteogenesis, mutations in BMP ligands or receptors have been identified as the basis of a wide range of skeletal disorders in humans.
Fibrodysplasia ossificans progressiva (FOP)
FOP is an extremely rare and debilitating genetic disorder characterized by congenital malformations of the halluces (big toes) and by progressive heterotopic endochondral ossifi- cation in predictable anatomical patterns. The classic pheno- type is caused by a mutation (617G > A; R206H) in ACVR1 that accounts for at least 98% of classic presentations [37].
Further studies have identified new mutations includ- ing c.982G > A (p.G328R), c.1124G > C (p.R375P), c.590_
592delCTT, P197_F198delinsL, and c.619C > G (Q207E) [37].
Recent studies have shown that these mutations in ACVR1 lead to altered responsiveness to activin. Activin ligands typi- cally bind to AVCR1 but do not induce signal transduction.
The FOP mutations lead to a structural alteration that ena- bles activin to activate BMP signaling through ACVR1 [38].
Thus, in addition to competition of activins and BMPs for type II receptors, the occupation of ACVR1 by either activin or BMP ligands appears to be a second mechanism by which BMP and activin pathways interact.
Osteoarthritis (OA)
OA is a disease involving degeneration of the articular cartilage in synovial joints, such as the knee, hip, and hand. Associations between polymorphisms in BMP5
Acknooledgments 65 and BMP2 and OA have been found, suggesting that vari-
ability in gene expression is a susceptibility factor for the disease. The strongest evidence for a role in OA suscepti- bility comes from a SNP in the 5’ untranslated region (5’‐UTR) of BMP14/GDF5, rs143383, associated with OA [39]. rs143383 is a C/T transition and the OA‐associated T allele of rs143383 has been shown to produce less mRNA transcript compared with the ancestral C allele, indicating that reduced BMP14/GDF5 expression is likely to be the mechanism through which this OA susceptibility locus is working.
Brachydactyly (BD)
BD is a shortening of the hands/feet due to small or missing metacarpals/metatarsals and/or phalanges. Depending on the affected phalanges, five different types of brachydactyl- ies are categorized (BDA to BDE) including seven subgroups (BDA1 to BDA7).
Mutations in BMP14/GDF5 have been linked to isolated traits of different types of brachydactyly including BDA1, BDA2, and BDC [40]. Dominant‐
negative mutations in BMPR1B and a specific missense mutation in BMP14/GDF5 are known to cause isolated brachydactyly type BDA2 due to a loss of interaction between BMPR1B and BMP‐14/GDF‐5 [41]. A mutation in BMPR1B (R486Q) is associated with either BDA2 or a BDC/symphalangism (SYM1)‐like phenotype [42].
Duplication of a regulatory element that affects the expression of BMP2 is associated with BDA2 [43].
Symphalangism (SYM)
SYM is an uncommon condition characterized by fusion of the joints of the fingers or toes. Activating mutations in BMP14/GDF5 result in increased chondrogenic activity as described for proximal SYM and the multiple synostoses syndrome 2 [44]. A mutation in BMP14 (R438L) leads to a loss of receptor‐binding specificity, causing the SYM phenotype [41].