ACVR1 p.Q207E causes classic fibrodysplasia ossificans progressiva and is functionally distinct from the engineered constitutively active ACVR1 p.Q207D variant.

Fibrodysplasia ossificans progressiva (FOP) is a disabling genetic disorder of progressive heterotopic ossification (HO). Here, we report a patient with an ultra-rare point mutation [c.619C>G, p.Q207E] located in a codon adjacent to the most common FOP mutation [c.617G>A, p.R206H] of Activin A Receptor, type 1 (ACVR1) and that affects the same intracellular amino acid position in the GS activation domain as the engineered constitutively active (c.a.) variant p.Q207D. It was predicted that both mutations at residue 207 have similar functional effects by introducing a negative charge. Transgenic p.Q207D-c.a. mice have served as a model for FOP HO in several in vivo studies. However, we found that the engineered ACVR1(Q207D-c.a.) is significantly more active than the classic FOP mutation ACVR1(R206H) when overexpressed in chicken limbs and in differentiation assays of chondrogenesis, osteogenesis and myogenesis. Importantly, our studies reveal that the ACVR1(Q207E) resembles the classic FOP receptor in these assays, not the engineered ACVR1(Q207D-c.a.). Notably, reporter gene assays revealed that both naturally occurring FOP receptors (ACVR1(R206H) and ACVR1(Q207E)) were activated by BMP7 and were sensitive to deletion of the ligand binding domain, whereas the engineered ACVR1(Q207D-c.a.) exhibited ligand independent activity. We performed an in silico analysis and propose a structural model for p.Q207D-c.a. that irreversibly relocates the GS domain into an activating position, where it becomes ligand independent. We conclude that the engineered p.Q207D-c.a. mutation has severe limitations as a model for FOP, whereas the naturally occurring mutations p.R206H and p.Q207E facilitate receptor activation, albeit in a reversible manner.

Mutations in GDF5 reveal a key residue mediating BMP inhibition by NOGGIN.

Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP-related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP-inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.

ActivinA Induced SMAD1/5 Signaling in an iPSC Derived EC Model of Fibrodysplasia Ossificans Progressiva (FOP) Can Be Rescued by the Drug Candidate Saracatinib.

Balanced signal transduction is crucial in tissue patterning, particularly in the vasculature. Heterotopic ossification (HO) is tightly linked to vascularization with increased vessel number in hereditary forms of HO, such as Fibrodysplasia ossificans progressiva (FOP). FOP is caused by mutations in the BMP type I receptor ACVR1 leading to aberrant SMAD1/5 signaling in response to ActivinA. Whether observed vascular phenotype in human FOP lesions is connected to aberrant ActivinA signaling is unknown. Blocking of ActivinA prevents HO in FOP mice indicating a central role of the ligand in FOP. Here, we established a new FOP endothelial cell model generated from induced pluripotent stem cells (iECs) to study ActivinA signaling. FOP iECs recapitulate pathogenic ActivinA/SMAD1/5 signaling. Whole transcriptome analysis identified ActivinA mediated activation of the BMP/NOTCH pathway exclusively in FOP iECs, which was rescued to WT transcriptional levels by the drug candidate Saracatinib. We propose that ActivinA causes transcriptional pre-patterning of the FOP endothelium, which might contribute to differential vascularity in FOP lesions compared to non-hereditary HO.

ACVR1R206H FOP mutation alters mechanosensing and tissue stiffness during heterotopic ossification.

An activating bone morphogenetic proteins (BMP) type I receptor ACVR1 (ACVR1R206H) mutation enhances BMP pathway signaling and causes the rare genetic disorder of heterotopic (extraskeletal) bone formation fibrodysplasia ossificans progressiva. Heterotopic ossification frequently occurs following injury as cells aberrantly differentiate during tissue repair. Biomechanical signals from the tissue microenvironment and cellular responses to these physical cues, such as stiffness and rigidity, are important determinants of cell differentiation and are modulated by BMP signaling. We used an Acvr1R206H/+ mouse model of injury-induced heterotopic ossification to examine the fibroproliferative tissue preceding heterotopic bone and identified pathologic stiffening at this stage of repair. In response to microenvironment stiffness, in vitro assays showed that Acvr1R206H/+ cells inappropriately sense their environment, responding to soft substrates with a spread morphology similar to wild-type cells on stiff substrates and to cells undergoing osteoblastogenesis. Increased activation of RhoA and its downstream effectors demonstrated increased mechanosignaling. Nuclear localization of the pro-osteoblastic factor RUNX2 on soft and stiff substrates suggests a predisposition to this cell fate. Our data support that increased BMP signaling in Acvr1R206H/+ cells alters the tissue microenvironment and results in misinterpretation of the tissue microenvironment through altered sensitivity to mechanical stimuli that lowers the threshold for commitment to chondro/osteogenic lineages.

Variable signaling activity by FOP ACVR1 mutations.

Most patients with fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder of heterotopic ossification, have the same causative mutation in ACVR1, R206H. However, additional mutations within the ACVR1 BMP type I receptor have been identified in a small number of FOP cases, often in patients with disease of lesser or greater severity than occurs with R206H mutations. Genotype-phenotype correlations have been suggested in patients, resulting in classification of FOP mutations based on location within different receptor domains and structural modeling. However while each of the mutations induces increased signaling through the BMP-pSmad1/5/8 pathway, the molecular mechanisms underlying functional differences of these FOP variant receptors remained undetermined. We now demonstrate that FOP mutations within the ACVR1 receptor kinase domain are more sensitive to low levels of BMP than mutations in the ACVR1 GS domain. Our data additionally confirm responsiveness of cells with FOP ACVR1 mutations to both BMP and Activin A ligands. We also have determined that constructs with FOP ACVR1 mutations that are engineered without the ligand-binding domain retain increased BMP-pSmad1/5/8 pathway activation relative to wild-type ACVR1, supporting that the mutant receptors can function through ligand-independent mechanisms either directly through mutant ACVR1 or through indirect mechanisms.

TGF-ß Family Signaling in Connective Tissue and Skeletal Diseases.

The transforming growth factor ß (TGF-ß) family of signaling molecules, which includes TGF-ßs, activins, inhibins, and numerous bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs), has important functions in all cells and tissues, including soft connective tissues and the skeleton. Specific TGF-ß family members play different roles in these tissues, and their activities are often balanced with those of other TGF-ß family members and by interactions with other signaling pathways. Perturbations in TGF-ß family pathways are associated with numerous human diseases with prominent involvement of the skeletal and cardiovascular systems. This review focuses on the role of this family of signaling molecules in the pathologies of connective tissues that manifest in rare genetic syndromes (e.g., syndromic presentations of thoracic aortic aneurysm), as well as in more common disorders (e.g., osteoarthritis and osteoporosis). Many of these diseases are caused by or result in pathological alterations of the complex relationship between the TGF-ß family of signaling mediators and the extracellular matrix in connective tissues.

Comparative expression pattern of Odd-skipped related genes Osr1 and Osr2 in chick embryonic development.

Odd-skipped genes encode zinc-finger transcription factors with widespread roles in embryonic development. In Drosophila, odd-skipped acts as a pair-rule gene, while its orthologous gene in Caenorhabditis elegans is involved in gut development. In mammals two paralogs exist, Osr1 and Osr2, with functions described in heart and urogenital, and in secondary palate development, respectively. As the chicken embryo is a widely used system for analysing gene function in vivo, we determined the expression pattern of the two chicken orthologues, cOsr1 and cOsr2, during embryonic development. We demonstrate expression of both genes in a variety of organs and structures, such as kidney, eye, branchial arches and dermis. Both genes show a highly dynamic expression pattern with partially overlapping, but mostly distinct domains of expression. Special emphasis in this study was laid on the investigation of cOsr1 and cOsr2 in limb development, where we compared their expression pattern with the expression of Osr1 and Osr2 in the mouse.

Odd-skipped related genes regulate differentiation of embryonic limb mesenchyme and bone marrow mesenchymal stromal cells.

The regulation of progenitor cell differentiation to a specific tissue type is one of the fundamental questions of biology. Here, we identify Osr1 and Osr2, 2 closely related genes encoding zinc finger transcription factors, as being strongly expressed in irregular connective tissue (ICT) fibroblasts in the chicken embryo, suitable as a developmental marker. We provide evidence that both Osr1 and Osr2 regulate mesenchymal cell-type differentiation. Both Osr1 and Osr2 can promote the formation of ICT, a cell type of so far unknown molecular specification, while suppressing differentiation of other tissues such as cartilage and tendon from uncommitted progenitors. Conversely, knockdown of either Osr gene alone or in combination reverses this effect, thereby leading to decreased differentiation of ICT fibroblasts and increased chondrogenesis in vitro. This indicates that Osr genes play a pivotal role in ICT fibroblast differentiation. Undifferentiated mesenchymal cells reside in the adult body in the form of mesenchymal stem cells in the bone marrow cavity. Using bone marrow stromal cells (BMSCs) isolated from chicken fetal long bones, we show that Osr1 and Osr2 have an intrinsic role in BMSC differentiation similar to their role in early embryonic development, that is, the enforcement of CT fibroblast differentiation and the repression of other cell types as exemplified here by osteoblast differentiation.

Investigations of activated ACVR1/ALK2, a bone morphogenetic protein type I receptor, that causes fibrodysplasia ossificans progressiva.

Bone morphogenetic protein (BMP) type I receptors are serine-threonine kinase transmembrane signal transduction proteins that regulate a vast array of ligand-dependent cell-fate decisions with temporal and spatial fidelity during development and postnatal life. A recent discovery identified a recurrent activating heterozygous missense mutation in a BMP type I receptor [Activin receptor IA/activin-like kinase 2 (ACVR1; also known as ALK2)] in patients with the disabling genetic disorder fibrodysplasia ossificans progressiva (FOP). Individuals with FOP experience episodes of tissue metamorphosis that convert soft connective tissue such as skeletal muscle into a highly ramified and disabling second skeleton of heterotopic bone. The single nucleotide ACVR1/ALK2 mutation that causes FOP is one of the most specific disease-causing mutations in the human genome and to date the only known inherited activating mutation of a BMP receptor that causes a human disease. Thus, the study of FOP provides the basis for understanding the clinically relevant effects of activating mutations in the BMP signaling pathway. Here we briefly review methodologies that we have applied to studying activated BMP signaling in FOP.

Homeobox genes d11-d13 and a13 control mouse autopod cortical bone and joint formation.

The molecular mechanisms that govern bone and joint formation are complex, involving an integrated network of signaling pathways and gene regulators. We investigated the role of Hox genes, which are known to specify individual segments of the skeleton, in the formation of autopod limb bones (i.e., the hands and feet) using the mouse mutant synpolydactyly homolog (spdh), which encodes a polyalanine expansion in Hoxd13. We found that no cortical bone was formed in the autopod in spdh/spdh mice; instead, these bones underwent trabecular ossification after birth. Spdh/spdh metacarpals acquired an ovoid shape and developed ectopic joints, indicating a loss of long bone characteristics and thus a transformation of metacarpals into carpal bones. The perichondrium of spdh/spdh mice showed abnormal morphology and decreased expression of Runt-related transcription factor 2 (Runx2), which was identified as a direct Hoxd13 transcriptional target. Hoxd11-/-Hoxd12-/-Hoxd13-/- triple-knockout mice and Hoxd13-/-Hoxa13+/- mice exhibited similar but less severe defects, suggesting that these Hox genes have similar and complementary functions and that the spdh allele acts as a dominant negative. This effect was shown to be due to sequestration of other polyalanine-containing transcription factors by the mutant Hoxd13 in the cytoplasm, leading to their degradation. These data indicate that Hox genes not only regulate patterning but also directly influence bone formation and the ossification pattern of bones, in part via Runx2.

The fibrodysplasia ossificans progressiva R206H ACVR1 mutation activates BMP-independent chondrogenesis and zebrafish embryo ventralization.

Patients with classic fibrodysplasia ossificans progressiva, a disorder characterized by extensive extraskeletal endochondral bone formation, share a recurrent mutation (R206H) within the glycine/serine-rich domain of ACVR1/ALK2, a bone morphogenetic protein type I receptor. Through a series of in vitro assays using several mammalian cell lines and chick limb bud micromass cultures, we determined that mutant R206H ACVR1 activated BMP signaling in the absence of BMP ligand and mediated BMP-independent chondrogenesis that was enhanced by BMP. We further investigated the interaction of mutant R206H ACVR1 with FKBP1A, a glycine/serine domain-binding protein that prevents leaky BMP type I receptor activation in the absence of ligand. The mutant protein exhibited reduced binding to FKBP1A in COS-7 simian kidney cell line assays, suggesting that increased BMP pathway activity in COS-7 cells with R206H ACVR1 is due, at least in part, to decreased binding of this inhibitory factor. Consistent with these findings, in vivo analyses of zebrafish embryos showed BMP-independent hyperactivation of BMP signaling in response to the R206H mutant, resulting in increased embryonic ventralization. These data support the conclusion that the mutant R206H ACVR1 receptor in FOP patients is an activating mutation that induces BMP signaling in a BMP-independent and BMP-responsive manner to promote chondrogenesis, consistent with the ectopic endochondral bone formation in these patients.