A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

Growth and Differentiation Factor 5 (GDF5) is a secreted growth factor that belongs to the Bone Morphogenetic Protein (BMP) family and plays a pivotal role during limb development. GDF5 is a susceptibility gene for osteoarthritis (OA) and mutations in GDF5 are associated with a wide variety of skeletal malformations ranging from complex syndromes such as acromesomelic chondrodysplasias to isolated forms of brachydactylies or multiple synostoses syndrome 2 (SYNS2). Here, we report on a family with an autosomal dominant inherited combination of SYNS2 and additional brachydactyly type A1 (BDA1) caused by a single point mutation in GDF5 (p.W414R). Functional studies, including chondrogenesis assays with primary mesenchymal cells, luciferase reporter gene assays and Surface Plasmon Resonance analysis, of the GDF5^W414R variant in comparison to other GDF5 mutations associated with isolated BDA1 (p.R399C) or SYNS2 (p.E491K) revealed a dual pathomechanism characterized by a gain- and loss-of-function at the same time. On the one hand insensitivity to the main GDF5 antagonist NOGGIN (NOG) leads to a GDF5 gain of function and subsequent SYNS2 phenotype. Whereas on the other hand, a reduced signaling activity, specifically via the BMP receptor type IA (BMPR1A), is likely responsible for the BDA1 phenotype. These results demonstrate that one mutation in the overlapping interface of antagonist and receptor binding site in GDF5 can lead to a GDF5 variant with pathophysiological relevance for both, BDA1 and SYNS2 development. Consequently, our study assembles another part of the molecular puzzle of how loss and gain of function mutations in GDF5 affect bone development in hands and feet resulting in specific types of brachydactyly and SYNS2. These novel insights into the biology of GDF5 might also provide further clues on the pathophysiology of OA.     

Fibrodysplasia ossificans progressiva and associated osteochondroma of the coronoid process in a child

The article reports the occurrence of osteochondroma in a fibrodysplasia ossificans progressiva patient. A 5-year-old boy presented with limited mouth opening and firm swelling of the right zygomatic complex area. The boy had bilateral hallux valgus of the great toes and heterotopic endochondral ossification of facial and neck regions. Associated osteochondroma of the coronoid process and aggressive heterotopic ossification of masticatory and neck muscles were found in response to traumatic injuries. Natural and clinical histories of fibrodysplasia ossificans progressiva were reviewed. An early diagnosis and avoidance of factors that aggravate ossification are key factors in reducing the expected degree of physical disabilities of patients. An early recognition of congenital skeletal deformities, early detection of abnormal ossification, and awareness of the disease by the involved physicians are important factors in the early diagnosis of the disease and in reducing any unnecessary trauma. Bone scintigrams and CT scans are effective noninvasive tools for an early detection of ossification and for monitoring the progression of the disease. Further investigation of its pathogenesis at a molecular level is important to understand better the nature of the disease and to develop an effective treatment protocol.     

Elevated prostaglandin production in cultured cells from a patient with fibrodysplasia ossificans progressiva.

Prostaglandin E production was measured in cells cultured from both a fibromatoid lesion and a normal area of skin in a patient with fibrodysplasia ossificans progressiva. The synthesis of prostaglandin E-like material (iPGE) was ten- to twenty-fold greater in cells cultured from the patient's fibromatoid lesion than in fibroblasts obtained from her normal skin. Addition of indomethacin in vitro resulted in a greater than 95% reduction of iPGE production in both cell cultures. These observations appear to warrant further investigation in additional patients, with this disease.     

HSPG modulation of BMP signaling in fibrodysplasia ossificans progressiva cells

Cell surface heparan sulfate proteoglycans (HSPGs) play important roles in morphogen gradient formation and cell signaling. Bone morphogenetic protein (BMP) signaling is dysregulated in fibrodysplasia ossificans progressiva (FOP), a disabling disorder of progressive heterotopic bone formation. Here, we investigated the role of HSPG glycosaminoglycan (GAG) side chains on BMP signaling and found increased total and HSPG-specific GAG chain levels and dysregulation in HSPG modulation of BMP signaling in FOP lymphoblastoid cells (LCLs). Specifically, HSPG profiling demonstrated abundant mRNA and protein levels of glypican 1 and syndecan 4 on control and FOP LCLs, with elevated core protein levels on FOP cells. Targeted downregulation of glypican 1 core protein synthesis by siRNA enhanced BMP signaling in control and FOP cells, while reduction of syndecan 4-core protein synthesis decreased BMP signaling in control, but not FOP cells. These results suggest that FOP cells are resistant to the stimulatory effects of cell surface HSPG GAG chains, but are susceptible to the inhibitory effects, as shown by downregulation of glypican 1. These data support that HSPG modulation of BMP signaling is altered in cells from patients with FOP and that altered HSPG-related BMP signaling may play a role in the pathogenesis of the disease.     

Elevated prostaglandin production in culture cells from a patient with fibrodysplasia ossificans progressiva

Prostaglandin E production was measured in cells cultured from both a fibromatoid lesion and a normal area of skin in a patient with fibrodysplasia ossificans progressiva. The synthesis of prostaglandin E-like material (iPGE) was ten- to twenty-fold greater in cells cultured from the patient's fibromatoid lesion than in fibroblasts obtained from her normal skin. Addition of indomethacin in vitro resulted in a greater than 95% reduction in iPGE production in both cell cultures. These observations appear to warrant further investigation in additional patients, with this disease.     

Dynamics of BMP signaling in limb bud mesenchyme and polydactyly

Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin, which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9-expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis.     

Temtamy preaxial brachydactyly syndrome is caused by loss-of-function mutations in chondroitin synthase 1, a potential target of BMP signaling

Altered Bone Morphogenetic Protein (BMP) signaling leads to multiple developmental defects, including brachydactyly and deafness. Here we identify chondroitin synthase 1 (CHSY1) as a potential mediator of BMP effects. We show that loss of human CHSY1 function causes autosomal-recessive Temtamy preaxial brachydactyly syndrome (TPBS), mainly characterized by limb malformations, short stature, and hearing loss. After mapping the TPBS locus to chromosome 15q26-qterm, we identified causative mutations in five consanguineous TPBS families. In zebrafish, antisense-mediated chsy1 knockdown causes defects in multiple developmental processes, some of which are likely to also be causative in the etiology of TPBS. In the inner ears of zebrafish larvae, chsy1 is expressed similarly to the BMP inhibitor dan and in a complementary fashion to bmp2b. Furthermore, unrestricted Bmp2b signaling or loss of Dan activity leads to reduced chsy1 expression and, during epithelial morphogenesis, defects similar to those that occur upon Chsy1 inactivation, indicating that Bmp signaling affects inner-ear development by repressing chsy1. In addition, we obtained strikingly similar zebrafish phenotypes after chsy1 overexpression, which might explain why, in humans, brachydactyly can be caused by mutations leading either to loss or to gain of BMP signaling.     

A new mutation in the gene ROR2 causes brachydactyly type B1

Brachydactyly type B, an autosomal dominant disorder that is characterized by hypoplasia of the distal phalanges and nails, can be divided into brachydactyly type B1 (BDB1) and brachydactyly type B2 (BDB2). BDB1 is caused by mutations in the receptor tyrosine kinase gene ROR2, which maps to chromosome 9q22, whereas BDB2 is caused by point mutations in the bone morphogenetic protein antagonist NOGGIN. Here, we report a three-generation Chinese family with dominant inheritance of the BDB1 limb phenotype. Sequence analysis identified a novel heterozygous base deletion (c.1396–1398delAA) in the gene ROR2 in all affected family members. This new deletion is expected to produce a truncated Ror2 protein with a new polypeptide of 57 amino acids at the C-terminal.     

BMP and mTOR signaling in heterotopic ossification: Does their crosstalk provide therapeutic opportunities?

Heterotopic ossification (HO) refers to the pathological formation of ectopic bone in soft tissues, it occurs following severe trauma or in patients with a rare genetic disorder known as fibrodysplasia ossificans progressiva. The pathological process of HO formation is a two-step mechanism: inflammation and destruction of connective tissues, followed by bone formation. The latter is further subdivided into three stages: fibroproliferation/angiogenesis, chondrogenesis, and osteogenesis. Currently, therapeutic options for HO are limited. New potential therapeutics will most likely arise from a more detailed understanding of the signaling pathways implicated in each stage of ectopic bone formation and molecular targets that may be effective at both the early and late stages of HO. Bone morphogenetic protein (BMP) signaling is believed to play a key role in the overall HO process. Recently, the mammalian target of rapamycin (mTOR) signaling pathway has received attention as a critical pathway for chondrogenesis, osteogenesis, and HO. Inhibition of mTOR signaling has been shown to block trauma-induced and genetic HO. Intriguingly, recent studies have revealed crosstalk between mTOR and BMP signaling. Moreover, mTOR has emerged as a factor involved in the early hypoxic and inflammatory stages of HO. We will summarize the current knowledge of the roles of mTOR and BMP signaling in HO, with a particular focus on the crosstalk between mTOR and BMP signaling. We also discuss the activation of AMP activated protein kinase (AMPK) by the most widely used drug for type 2 diabetes, metformin, which exerts a dual negative regulatory effect on mTOR and BMP signaling, suggesting that metformin is a promising drug treatment for HO. The discovery of an mTOR-BMP signaling network may be a potential molecular mechanism of HO and may represent a novel therapeutic target for the pharmacological control of HO.     

Discovery,synthesis and characterization of a series of 7-aryl-imidazo[1,2-a]pyridine-3-ylquinolines as activin-like kinase (ALK) inhibitors

The activin-like kinases are a family of kinases that play important roles in a variety of disease states. Of this class of kinases, ALK2, has been shown by a gain-of-function to be the primary driver of the childhood skeletal disease fibrodysplasia ossificans progressiva (FOP) and more recently the pediatric cancer diffuse intrinsic pontine glioma (DIPG). Herein, we report our efforts to identify a novel imidazo[1,2-a]pyridine scaffold as potent inhibitors of ALK2 with good in vivo pharmacokinetic properties suitable for future animal studies.     

Nevoid basal cell carcinoma syndrome (Gorlin syndrome)

Brachydactyly ("short digits") is a general term that refers to disproportionately short fingers and toes, and forms part of the group of limb malformations characterized by bone dysostosis. The various types of isolated brachydactyly are rare, except for types A3 and D. Brachydactyly can occur either as an isolated malformation or as a part of a complex malformation syndrome. To date, many different forms of brachydactyly have been identified. Some forms also result in short stature. In isolated brachydactyly, subtle changes elsewhere may be present. Brachydactyly may also be accompanied by other hand malformations, such as syndactyly, polydactyly, reduction defects, or symphalangism. For the majority of isolated brachydactylies and some syndromic forms of brachydactyly, the causative gene defect has been identified. In isolated brachydactyly, the inheritance is mostly autosomal dominant with variable expressivity and penetrtance. Diagnosis is clinical, anthropometric and radiological. Prenatal diagnosis is usually not indicated for isolated forms of brachydactyly, but may be appropriate in syndromic forms. Molecular studies of chorionic villus samples at 11 weeks of gestation and by amniocentesis after the 14^th week of gestation can provide antenatal diagnosis if the causative mutation in the family is known. The nature of genetic counseling depends both on the pattern of inheritance of the type of brachydactyly present in the family and on the presence or absence of accompanying symptoms. There is no specific management or treatment that is applicable to all forms of brachydactyly. Plastic surgery is only indicated if the brachydactyly affects hand function or for cosmetic reasons, but is typically not needed. Physical therapy and ergotherapy may ameliorate hand function. Prognosis for the brachydactylies is strongly dependent on the nature of the brachydactyly, and may vary from excellent to severely influencing hand function. If brachydactyly forms part of a syndromic entity, prognosis often depends on the nature of the associated anomalies.     

The gene for bone morphogenetic protein 2A (BMP2A) is localized to human chromosome 20pl2 by radioactive and nonradioactive in situ hybridization

Bone morphogenetic protein 2A (BMP2A), a member of the decapentaplegic-Vg-related family, belongs to the transforming growth factor superfamily and has a striking sequence similarity to the decapentaplegic locus in Drosophila melanogaster, a major determinant of pattern specification during embryogenesis. BMP2A is thought to be involved in cartilage and bone formation during embryogenesis, but may have additional functions in morphogenesis as implied by its expression in various organs and embryonic tissues of mice. Human BMP2A, assigned to chromosome 20 by the use of humanChinese hamster ovary cell hybrids, is considered to be a reasonable candidate gene for the autosomal dominant disease of fibrodysplasia (myositis) ossificans progressiva. We have confirmed the localization of BMP2A to chromosome 20 and regionally assigned the locus to 20pl2 by radioactive and nonradioactive in situ hybridization.     

A new subtype of brachydactyly type B caused by point mutations in the bone morphogenetic protein antagonist NOGGIN

Brachydactyly type B (BDB) is characterized by terminal deficiency of fingers and toes, which is caused by heterozygous truncating mutations in the receptor tyrosine kinase-like orphan receptor 2 (ROR2) in the majority of patients. In a subset of ROR2-negative patients with BDB, clinically defined by the additional occurrence of proximal symphalangism and carpal synostosis, we identified six different point mutations (P35A, P35S, A36P, E48K, R167G, and P187S) in the bone morphogenetic protein (BMP) antagonist NOGGIN (NOG). In contrast to previously described loss-of-function mutations in NOG, which are known to cause a range of conditions associated with abnormal joint formation but without BDB, the newly identified BDB mutations do not indicate a major loss of function, as suggested by calculation of free-binding energy of the modeled NOG-GDF5 complex and functional analysis of the micromass culture system. Rather, they presumably alter NOG's ability to bind to BMPs and growth-differentiation factors (GDFs) in a subtle way, thus disturbing the intricate balance of BMP signaling. The combined features observed in this phenotypic subtype of BDB argue for a functional connection between BMP and ROR2 signaling and support previous findings of a modulating effect of ROR2 on the BMP-receptor pathway through the formation of a heteromeric complex of the receptors at the cell surface.     

Substance P signaling mediates BMP-dependent heterotopic ossification

Heterotopic ossification (HO) is a disabling condition associated with neurologic injury, inflammation, and overactive bone morphogenetic protein (BMP) signaling. The inductive factors involved in lesion formation are unknown. We found that the expression of the neuro-inflammatory factor Substance P (SP) is dramatically increased in early lesional tissue in patients who have either fibrodysplasia ossificans progressiva (FOP) or acquired HO, and in three independent mouse models of HO. In Nse-BMP4, a mouse model of HO, robust HO forms in response to tissue injury; however, null mutations of the preprotachykinin (PPT) gene encoding SP prevent HO. Importantly, ablation of SP(+) sensory neurons, treatment with an antagonist of SP receptor NK1r, deletion of NK1r gene, or genetic down-regulation of NK1r-expressing mast cells also profoundly inhibit injury-induced HO. These observations establish a potent neuro-inflammatory induction and amplification circuit for BMP-dependent HO lesion formation, and identify novel molecular targets for prevention of HO.     

Bone Morphogenetic Protein functions as a context-dependent angiogenic cue in vertebrates

Bone Morphogenetic Protein (BMP) signaling has been implicated in diverse biological processes. Although how BMP signaling regulates behaviors of endothelial cells during angiogenesis are not fully understood, increasing evidence indicate functions of BMP signaling components are essential in developmental and pathological angiogenesis. Here we review recent advances in delineating the functions of BMP signaling during angiogenesis. In addition, we discuss downstream pathways that transduce BMP signaling in endothelial cells, and factors that modulate BMP signaling response in endothelial cells. Finally, we provide recent insight on how BMP signaling functions as a context dependent angiogenic cue.     

Fibrodysplasia Ossificans Progressiva-related Activated Activin-like Kinase Signaling Enhances Osteoclast Formation during Heterotopic Ossification in Muscle Tissues

Fibrodysplasia ossificans progressiva is characterized by extensive ossification within muscle tissues, and its molecular pathogenesis is responsible for the constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2). In this study, we investigated the effects of implanting ALK2 (R206H)-transfected myoblastic C2C12 cells into nude mice on osteoclast formation during heterotopic ossification in muscle and subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells with BMP-2 in nude mice induced robust heterotopic ossification with an increase in the formation of osteoclasts in muscle tissues but not in subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells in muscle induced heterotopic ossification more effectively than that of empty vector-transfected cells. A co-culture of ALK2 (R206H)-transfected C2C12 cells as well as the conditioned medium from ALK2 (R206H)-transfected C2C12 cells enhanced osteoclast formation in Raw264.7 cells more effectively than those with empty vector-transfected cells. The transfection of ALK2 (R206H) into C2C12 cells elevated the expression of transforming growth factor (TGF)-β, whereas the inhibition of TGF-β signaling suppressed the enhanced formation of osteoclasts in the co-culture with ALK2 (R206H)-transfected C2C12 cells and their conditioned medium. In conclusion, this study demonstrated that the causal mutation transfection of fibrodysplasia ossificans progressiva in myoblasts enhanced the formation of osteoclasts from its precursor through TGF-β in muscle tissues.     

A new function of BMP4: dual role for BMP4 in regulation of Sonic hedgehog expression in the mouse tooth germ

The murine tooth development is governed by sequential and reciprocal epithelial-mesenchymal interactions. Multiple signaling molecules are expressed in the developing tooth germ and interact each other to mediate the inductive tissue interactions. Among them are Sonic hedgehog (SHH), Bone Morphogenetic Protein-2 (BMP2) and Bone Morphogenetic Protein-4 (BMP4). We have investigated the interactions between these signaling molecules during early tooth development. We found that the expression of Shh and Bmp2 is downregulated at E12.5 and E13.5 in the dental epithelium of the Msx1 mutant tooth germ where Bmp4 expression is significantly reduced in the dental mesenchyme. Inhibition of BMP4 activity by noggin resulted in repression of Shh and Bmp2 in wild-type dental epithelium. When implanted into the dental mesenchyme of Msx1 mutants, beads soaked with BMP4 protein were able to restore the expression of both Shh and Bmp2 in the Msx1 mutant epithelium. These results demonstrated that mesenchymal BMP4 represents one component of the signal acting on the epithelium to maintain Shh and Bmp2 expression. In contrast, BMP4-soaked beads repressed Shh and Bmp2 expression in the wild-type dental epithelium. TUNEL assay indicated that this suppression of gene expression by exogenous BMP4 was not the result of an increase in programmed cell death in the tooth germ. Ectopic expression of human Bmp4 to the dental mesenchyme driven by the mouse Msx1 promoter restored Shh expression in the Msx1 mutant dental epithelium but repressed Shh in the wild-type tooth germ in vivo. We further demonstrated that this regulation of Shh expression by BMP4 is conserved in the mouse developing limb bud. In addition, Shh expression was unaffected in the developing limb buds of the transgenic mice in which a constitutively active Bmpr-IB is ectopically expressed in the forelimb posterior mesenchyme and throughout the hindlimb mesenchyme, suggesting that the repression of Shh expression by BMP4 may not be mediated by BMP receptor-IB. These results provide evidence for a new function of BMP4. BMP4 can act upstream to Shh by regulating Shh expression in mouse developing tooth germ and limb bud. Taken together, our data provide insight into a new regulatory mechanism for Shh expression, and suggest that this BMP4-mediated pathway in Shh regulation may have a general implication in vertebrate organogenesis.