RESUMEN
Fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder of progressive extra-skeletal ossification, is the most disabling form of heterotopic ossification (HO) in humans. Most people with FOP carry an activating mutation in a BMP type I receptor gene, ACVR1 R206H, that promotes ectopic chondrogenesis and osteogenesis and in turn HO. Advances in elucidating the cellular and molecular events and mechanisms that lead to the ectopic bone formation are being made through the use of genetically engineered mouse models that recapitulate the human disease. We describe methods for inducing heterotopic ossification in a mouse model that conditionally expresses the Acvr1 R206H allele.
Asunto(s)
Miositis Osificante/patología , Osificación Heterotópica/patología , Receptores de Activinas Tipo I/genética , Alelos , Animales , Biomarcadores , Cardiotoxinas/efectos adversos , Modelos Animales de Enfermedad , Inmunohistoquímica , Ratones , Ratones Transgénicos , Mutación , Miositis Osificante/diagnóstico por imagen , Miositis Osificante/etiología , Osificación Heterotópica/diagnóstico por imagen , Osificación Heterotópica/etiología , Microtomografía por Rayos XRESUMEN
Inherited point mutations in collagen II in humans affecting mainly cartilage are broadly classified as chondrodysplasias. Most mutations occur in the glycine (Gly) of the Gly-X-Y repeats leading to destabilization of the triple helix. Arginine to cysteine substitutions that occur at either the X or Y position within the Gly-X-Y cause different phenotypes like Stickler syndrome and congenital spondyloepiphyseal dysplasia (SEDC). We investigated the consequences of arginine to cysteine substitutions (X or Y position within the Gly-X-Y) towards the N and C terminus of the triple helix. Protein expression and its secretion trafficking were analyzed. Substitutions R75C, R134C and R704C did not alter the thermal stability with respect to wild type; R740C and R789C proteins displayed significantly reduced melting temperatures (Tm) affecting thermal stability. Additionally, R740C and R789C were susceptible to proteases; in cell culture, R789C protein was further cleaved by matrix metalloproteinases (MMPs) resulting in expression of only a truncated fragment affecting its secretion and intracellular retention. Retention of misfolded R740C and R789C proteins triggered an ER stress response leading to apoptosis of the expressing cells. Arginine to cysteine mutations towards the C-terminus of the triple helix had a deleterious effect, whereas mutations towards the N-terminus of the triple helix (R75C and R134C) and R704C had less impact.
Asunto(s)
Sustitución de Aminoácidos , Colágeno Tipo II/genética , Osteocondrodisplasias/congénito , Respuesta de Proteína Desplegada , Línea Celular Tumoral , Supervivencia Celular , Colágeno Tipo II/química , Colágeno Tipo II/metabolismo , Células HEK293 , Humanos , Osteocondrodisplasias/genética , Desnaturalización Proteica , Dominios Proteicos , Estabilidad Proteica , Transporte de ProteínasAsunto(s)
Miositis Osificante , Osificación Heterotópica , Animales , Modelos Animales de Enfermedad , Macrófagos , Mastocitos , RatonesRESUMEN
Heterotopic ossification (HO) is a clinical condition that often reduces mobility and diminishes quality of life for affected individuals. The most severe form of progressive HO occurs in those with fibrodysplasia ossificans progressiva (FOP; OMIM #135100), a genetic disorder caused by a recurrent heterozygous gain-of-function mutation (R206H) in the bone morphogenetic protein (BMP) type I receptor ACVR1/ALK2. In individuals with FOP, episodes of HO frequently follow injury. The first sign of active disease is commonly an inflammatory "flare-up" that precedes connective tissue degradation, progenitor cell recruitment, and endochondral HO. We used a conditional-on global knock-in mouse model expressing Acvr1R206H (referred to as Acvr1cR206H/+ ) to investigate the cellular and molecular inflammatory response in FOP lesions following injury. We found that the Acvr1 R206H mutation caused increased BMP signaling in posttraumatic FOP lesions and early divergence from the normal skeletal muscle repair program with elevated and prolonged immune cell infiltration. The proinflammatory cytokine response of TNFα, IL-1ß, and IL-6 was elevated and prolonged in Acvr1cR206H/+ lesions and in Acvr1cR206H/+ mast cells. Importantly, depletion of mast cells and macrophages significantly impaired injury-induced HO in Acvr1cR206H/+ mice, reducing injury-induced HO volume by â¼50% with depletion of each cell population independently, and â¼75% with combined depletion of both cell populations. Together, our data show that the immune system contributes to the initiation and development of HO in FOP. Further, the expression of Acvr1R206H in immune cells alters cytokine expression and cellular response to injury and unveils novel therapeutic targets for treatment of FOP and nongenetic forms of HO. © 2017 American Society for Bone and Mineral Research.
Asunto(s)
Receptores de Activinas Tipo I/genética , Macrófagos/patología , Mastocitos/patología , Miositis Osificante/patología , Osificación Heterotópica/patología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Recuento de Células , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Mastocitos/metabolismo , Ratones , Músculo Esquelético/patología , Mutación/genética , Osificación Heterotópica/metabolismo , Transducción de SeñalRESUMEN
Fibrodysplasia ossificans progressiva (FOP), a rare and as yet untreatable genetic disorder of progressive extraskeletal ossification, is the most disabling form of heterotopic ossification (HO) in humans and causes skeletal deformities, movement impairment, and premature death. Most FOP patients carry an activating mutation in a bone morphogenetic protein (BMP) type I receptor gene, ACVR1(R206H) , that promotes ectopic chondrogenesis and osteogenesis and, in turn, HO. We showed previously that the retinoic acid receptor γ (RARγ) agonist palovarotene effectively inhibited HO in injury-induced and genetic mouse models of the disease. Here we report that the drug additionally prevents spontaneous HO, using a novel conditional-on knock-in mouse line carrying the human ACVR1(R206H) mutation for classic FOP. In addition, palovarotene restored long bone growth, maintained growth plate function, and protected growing mutant neonates when given to lactating mothers. Importantly, palovarotene maintained joint, limb, and body motion, providing clear evidence for its encompassing therapeutic potential as a treatment for FOP. © 2016 American Society for Bone and Mineral Research.
Asunto(s)
Receptores de Activinas Tipo I/genética , Extremidades/crecimiento & desarrollo , Extremidades/fisiopatología , Mutación/genética , Miositis Osificante/genética , Osificación Heterotópica/tratamiento farmacológico , Osificación Heterotópica/fisiopatología , Pirazoles/uso terapéutico , Estilbenos/uso terapéutico , Animales , Huesos/anomalías , Huesos/patología , Proliferación Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Condrocitos/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Placa de Crecimiento/efectos de los fármacos , Placa de Crecimiento/patología , Homeostasis , Humanos , Ratones Transgénicos , Movimiento , Osificación Heterotópica/patología , Osteogénesis , Pirazoles/farmacología , Estilbenos/farmacologíaRESUMEN
Bone morphogenetic protein (BMP) signaling is a critical regulator of cartilage differentiation and endochondral ossification. Gain-of-function mutations in ALK2, a type I BMP receptor, cause the debilitating disorder fibrodysplasia ossificans progressiva (FOP) and result in progressive heterotopic (extraskeletal) endochondral ossification within soft connective tissues. Here, we used murine mesenchymal progenitor cells to investigate the contribution of Alk2 during chondrogenic differentiation and heterotopic endochondral ossification (HEO). Alk2(R206H/+) (gain-of-function), Alk2(CKO) (loss-of-function), and wild-type mouse embryonic fibroblasts were evaluated for chondrogenic potential. Chondrogenic differentiation was accelerated in Alk2(R206H/+) cells, due in part to enhanced sensitivity to BMP ligand. In vivo, Alk2(R206H/+) cells initiated robust HEO and recruited wild-type cell contribution. Despite expression of other type I BMP receptors (Alk3 and Alk6), chondrogenesis of Alk2(CKO) cells was severely impaired by absence of Alk2 during early differentiation. Alk2 is therefore a direct regulator of cartilage formation and mediates chondrogenic commitment of progenitor cells. These data establish that at least one effect of ALK2 gain-of-function mutations in FOP patients is enhanced chondrogenic differentiation which supports formation of heterotopic endochondral bone. This establishes ALK2 as a plausible therapeutic target during early chondrogenic stages of lesion formation for preventing heterotopic bone formation in FOP and other conditions.
Asunto(s)
Receptores de Activinas Tipo I/genética , Condrogénesis/genética , Miositis Osificante/genética , Osificación Heterotópica/genética , Receptores de Activinas Tipo I/metabolismo , Animales , Proteína Morfogenética Ósea 4/farmacología , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Condrogénesis/efectos de los fármacos , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Immunoblotting , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Miositis Osificante/metabolismo , Miositis Osificante/patología , Osificación Heterotópica/metabolismo , Osificación Heterotópica/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de TiempoRESUMEN
Fibrodysplasia ossificans progressiva (FOP; MIM #135100) is a debilitating genetic disorder of connective tissue metamorphosis. It is characterized by malformation of the great (big) toes during embryonic skeletal development and by progressive heterotopic endochondral ossification (HEO) postnatally, which leads to the formation of a second skeleton of heterotopic bone. Individuals with these classic clinical features of FOP have the identical heterozygous activating mutation (c.617G>A; R206H) in the gene encoding ACVR1 (also known as ALK2), a bone morphogenetic protein (BMP) type I receptor. Disease activity caused by this ACVR1 mutation also depends on altered cell and tissue physiology that can be best understood in the context of a high-fidelity animal model. Recently, we developed such a knock-in mouse model for FOP (Acvr1(R206H/+)) that recapitulates the human disease, and provides a valuable new tool for testing and developing effective therapies. The FOP knock-in mouse and other models in Drosophila, zebrafish, chickens and mice provide an arsenal of tools for understanding BMP signaling and addressing outstanding questions of disease mechanisms that are relevant not only to FOP but also to a wide variety of disorders associated with regenerative medicine and tissue metamorphosis.
Asunto(s)
Desarrollo Óseo , Modelos Animales de Enfermedad , Metamorfosis Biológica , Miositis Osificante/patología , Miositis Osificante/fisiopatología , Receptores de Activinas/genética , Animales , Humanos , Mutación/genética , Miositis Osificante/genética , Miositis Osificante/terapiaRESUMEN
Fibrodysplasia ossificans progressiva (FOP; MIM #135100) is a debilitating genetic disorder of dysregulated cellular differentiation characterized by malformation of the great toes during embryonic skeletal development and by progressive heterotopic endochondral ossification postnatally. Patients with these classic clinical features of FOP have the identical heterozygous single nucleotide substitution (c.617G > A; R206H) in the gene encoding ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor. Gene targeting was used to develop an Acvr1 knock-in model for FOP (Acvr1(R206H/+)). Radiographic analysis of Acvr1(R206H/+) chimeric mice revealed that this mutation induced malformed first digits in the hind limbs and postnatal extraskeletal bone formation, recapitulating the human disease. Histological analysis of murine lesions showed inflammatory infiltration and apoptosis of skeletal muscle followed by robust formation of heterotopic bone through an endochondral pathway, identical to that seen in patients. Progenitor cells of a Tie2(+) lineage participated in each stage of endochondral osteogenesis. We further determined that both wild-type (WT) and mutant cells are present within the ectopic bone tissue, an unexpected finding that indicates that although the mutation is necessary to induce the bone formation process, the mutation is not required for progenitor cell contribution to bone and cartilage. This unique knock-in mouse model provides novel insight into the genetic regulation of heterotopic ossification and establishes the first direct in vivo evidence that the R206H mutation in ACVR1 causes FOP.