RESUMO
Single case studies of extraordinary disease resilience may provide therapeutic insight into conditions for which no definitive treatments exist. An otherwise healthy 35-year-old man (patient-R) with the canonical pathogenic ACVR1R206H variant and the classic congenital great toe malformation of fibrodysplasia ossificans progressiva (FOP) had extreme paucity of post-natal heterotopic ossification (HO) and nearly normal mobility. We hypothesized that patient-R lacked a sufficient post-natal inflammatory trigger for HO. A plasma biomarker survey revealed a reduction in total matrix metalloproteinase-9 (MMP-9) compared to healthy controls and individuals with quiescent FOP. Whole exome sequencing identified compound heterozygous variants in MMP-9 (c.59C > T, p.A20V and c.493G > A, p.D165N). Structural analysis of the D165N variant predicted both decreased MMP-9 secretion and activity that were confirmed by enzyme-linked immunosorbent assay and gelatin zymography. Further, human proinflammatory M1-like macrophages expressing either MMP-9 variant produced significantly less Activin A, an obligate ligand for HO in FOP, compared to wildtype controls. Importantly, MMP-9 inhibition by genetic, biologic, or pharmacologic means in multiple FOP mouse models abrogated trauma-induced HO, sequestered Activin A in the extracellular matrix (ECM), and induced regeneration of injured skeletal muscle. Our data suggest that MMP-9 is a druggable node linking inflammation to HO, orchestrates an existential role in the pathogenesis of FOP, and illustrates that a single patient's clinical phenotype can reveal critical molecular mechanisms of disease that unveil novel treatment strategies.
A healthy 35-year-old man (patient-R) with the classic fibrodysplasia ossificans progressiva (FOP) mutation and the congenital great toe malformation of FOP had extreme lack of heterotopic ossification (HO) and nearly normal mobility. We hypothesized that patient-R lacked a sufficient inflammatory trigger for HO. Blood tests revealed a reduction in the level of an inflammatory protein called matrix metalloproteinase-9 (MMP-9) compared to other individuals with FOP as well as healthy controls. DNA analysis in patient-R identified mutations in MMP-9, one of which predicted decreased activity of MMP-9 which was confirmed by further testing. Inflammatory cells (macrophages) expressing the MMP-9 mutations identified in patient-R produced significantly less Activin A, an obligate stimulus for HO in FOP. In order to determine if MMP-9 deficiency was a cause of HO prevention in FOP, we inhibited MMP-9 activity by genetic, biologic, or pharmacologic means in FOP mouse models and showed that MMP-9 inhibition prevented or dramatically decreased trauma-induced HO in FOP, locked-up Activin A in the extracellular matrix, and induced regeneration of injured skeletal muscle. Our data show that MMP-9 links inflammation to HO and illustrate that one patient's clinical picture can reveal critical molecular mechanisms of disease that unveil new treatment strategies.
Assuntos
Receptores de Ativinas Tipo I , Metaloproteinase 9 da Matriz , Miosite Ossificante , Adulto , Animais , Humanos , Masculino , Camundongos , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Receptores de Ativinas Tipo I/deficiência , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Miosite Ossificante/genética , Miosite Ossificante/patologia , Miosite Ossificante/metabolismo , Ossificação Heterotópica/patologia , Ossificação Heterotópica/genética , Ossificação Heterotópica/metabolismoRESUMO
Skeletal bone formation and maintenance requires coordinate functions of several cell types, including bone forming osteoblasts and bone resorbing osteoclasts. Gsα, the stimulatory subunit of heterotrimeric G proteins, activates downstream signaling through cAMP and plays important roles in skeletal development by regulating osteoblast differentiation. Here, we demonstrate that Gsα signaling also regulates osteoclast differentiation during bone modeling and remodeling. Gnas, the gene encoding Gsα, is imprinted. Mice with paternal allele deletion of Gnas (Gnas+/p-) have defects in cortical bone quality and strength during early development (bone modeling) that persist during adult bone remodeling. Reduced bone quality in Gnas+/p- mice was associated with increased endosteal osteoclast numbers, with no significant effects on osteoblast number and function. Osteoclast differentiation and resorption activity was enhanced in Gnas+/p- cells. During differentiation, Gnas+/p- cells showed diminished pCREB, ß-catenin and cyclin D1, and enhanced Nfatc1 levels, conditions favoring osteoclastogenesis. Forskolin treatment increased pCREB and rescued osteoclast differentiation in Gnas+/p- by reducing Nfatc1 levels. Cortical bone of Gnas+/p- mice showed elevated expression of Wnt inhibitors sclerostin and Sfrp4 consistent with reduced Wnt/ß-catenin signaling. Our data identify a new role for Gsα signaling in maintaining bone quality by regulating osteoclast differentiation and function through cAMP/PKA and Wnt/ß-catenin pathways.
Assuntos
Diferenciação Celular , Cromograninas/metabolismo , Osso Cortical/citologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Osteoclastos/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Cromograninas/genética , Osso Cortical/metabolismo , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclina D1/metabolismo , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Hypoxia and inflammation are implicated in the episodic induction of heterotopic endochondral ossification (HEO); however, the molecular mechanisms are unknown. HIF-1α integrates the cellular response to both hypoxia and inflammation and is a prime candidate for regulating HEO. We investigated the role of hypoxia and HIF-1α in fibrodysplasia ossificans progressiva (FOP), the most catastrophic form of HEO in humans. We found that HIF-1α increases the intensity and duration of canonical bone morphogenetic protein (BMP) signaling through Rabaptin 5 (RABEP1)-mediated retention of Activin A receptor, type I (ACVR1), a BMP receptor, in the endosomal compartment of hypoxic connective tissue progenitor cells from patients with FOP. We further show that early inflammatory FOP lesions in humans and in a mouse model are markedly hypoxic, and inhibition of HIF-1α by genetic or pharmacologic means restores canonical BMP signaling to normoxic levels in human FOP cells and profoundly reduces HEO in a constitutively active Acvr1(Q207D/+) mouse model of FOP. Thus, an inflammation and cellular oxygen-sensing mechanism that modulates intracellular retention of a mutant BMP receptor determines, in part, its pathologic activity in FOP. Our study provides critical insight into a previously unrecognized role of HIF-1α in the hypoxic amplification of BMP signaling and in the episodic induction of HEO in FOP and further identifies HIF-1α as a therapeutic target for FOP and perhaps nongenetic forms of HEO. © 2016 American Society for Bone and Mineral Research.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Ossificação Heterotópica/metabolismo , Ossificação Heterotópica/patologia , Transdução de Sinais , Receptores de Ativinas Tipo I/metabolismo , Animais , Hipóxia Celular , Condrogênese , Modelos Animais de Doenças , Endossomos/metabolismo , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/patologia , Ligantes , Masculino , Camundongos Transgênicos , Modelos Biológicos , Miosite Ossificante/patologia , Células-Tronco/metabolismo , Células-Tronco/patologia , Esfoliação de Dente/patologia , Dente Decíduo/patologiaRESUMO
The most important milestone in understanding a genetic disease is the identification of the causative mutation. However, such knowledge is often insufficient to decipher the pathophysiology of the disorder or to effectively treat those affected. Fibrodysplasia ossificans progressiva (FOP) is a rare, disabling, genetic disease of progressive heterotopic endochondral ossification (HEO) enabled by missense mutations that promiscuously and provisionally activate ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor, in all affected individuals. While activating mutations of the ACVR1/ALK2 receptor are necessary, disease activity and progression also depend on altered cell and tissue physiology. Recent findings identify inflammatory and immunological factors, vascular-derived mesenchymal stem cells, and a hypoxic lesional microenvironment that trigger, promote, and enable episodic progression of FOP in the setting of the genetic mutation. Effective therapies for FOP will need to consider these seminal pathophysiologic interactions.
Assuntos
Miosite Ossificante/genética , Miosite Ossificante/terapia , Receptores de Ativinas Tipo I/genética , Animais , Humanos , Mutação de Sentido Incorreto/genética , Miosite Ossificante/diagnóstico , Transplante de Células-Tronco/métodos , Células-Tronco/fisiologiaRESUMO
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.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Ossificação Heterotópica/metabolismo , Substância P/metabolismo , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Feminino , Humanos , Imuno-Histoquímica , Isoindóis/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Miosite Ossificante/genética , Miosite Ossificante/metabolismo , Antagonistas dos Receptores de Neurocinina-1 , Ossificação Heterotópica/genética , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Receptores da Neurocinina-1/metabolismo , Células Receptoras Sensoriais/metabolismo , Taquicininas/genética , Taquicininas/metabolismoRESUMO
Mesenchymal stem cells can give rise to several cell types, but varying results depending on isolation methods and tissue source have led to controversies about their usefulness in clinical medicine. Here we show that vascular endothelial cells can transform into multipotent stem-like cells by an activin-like kinase-2 (ALK2) receptor-dependent mechanism. In lesions from individuals with fibrodysplasia ossificans progressiva (FOP), a disease in which heterotopic ossification occurs as a result of activating ALK2 mutations, or from transgenic mice expressing constitutively active ALK2, chondrocytes and osteoblasts expressed endothelial markers. Lineage tracing of heterotopic ossification in mice using a Tie2-Cre construct also suggested an endothelial origin of these cell types. Expression of constitutively active ALK2 in endothelial cells caused endothelial-to-mesenchymal transition and acquisition of a stem cell-like phenotype. Similar results were obtained by treatment of untransfected endothelial cells with the ligands transforming growth factor-ß2 (TGF-ß2) or bone morphogenetic protein-4 (BMP4) in an ALK2-dependent manner. These stem-like cells could be triggered to differentiate into osteoblasts, chondrocytes or adipocytes. We suggest that conversion of endothelial cells to stem-like cells may provide a new approach to tissue engineering.
Assuntos
Receptores de Ativinas Tipo I/metabolismo , Diferenciação Celular/fisiologia , Células Endoteliais/citologia , Células-Tronco Multipotentes/citologia , Miosite Ossificante/metabolismo , Osteogênese/fisiologia , Engenharia Tecidual/métodos , Receptores de Ativinas Tipo I/genética , Animais , Proteína Morfogenética Óssea 4/metabolismo , Linhagem da Célula , Células Cultivadas , Células Endoteliais/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Transgênicos , Células-Tronco Multipotentes/metabolismo , Mutação/genética , Miosite Ossificante/genética , Oligonucleotídeos/genética , Interferência de RNA , Medicina Regenerativa/métodos , Fator de Crescimento Transformador beta2/metabolismoRESUMO
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.
Assuntos
Receptores de Ativinas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Miosite Ossificante/metabolismo , Receptores de Ativinas Tipo I/genética , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Análise Mutacional de DNA , Humanos , Immunoblotting , Imunoprecipitação , Miosite Ossificante/genéticaRESUMO
BACKGROUND: Individuals who have fibrodysplasia ossificans progressiva develop an ectopic skeleton because of genetic dysregulation of bone morphogenetic protein (BMP) signaling in the presence of inflammatory triggers. The identity of progenitor cells that contribute to various stages of BMP-induced heterotopic ossification relevant to fibrodysplasia ossificans progressiva and related disorders is unknown. An understanding of the cellular basis of heterotopic ossification will aid in the development of targeted, cell-specific therapies for the treatment and prevention of heterotopic ossification. METHODS: We used Cre/loxP lineage tracing methods in the mouse to identify cell lineages that contribute to all stages of heterotopic ossification. Specific cell populations were permanently labeled by crossing lineage-specific Cre mice with the Cre-dependent reporter mice R26R and R26R-EYFP. Two mouse models were used to induce heterotopic ossification: (1) intramuscular injection of BMP2/Matrigel and (2) cardiotoxin-induced skeletal muscle injury in transgenic mice that misexpress BMP4 at the neuromuscular junction. The contribution of labeled cells to fibroproliferative lesions, cartilage, and bone was evaluated histologically by light and fluorescence microscopy. The cell types evaluated as possible progenitors included skeletal muscle stem cells (MyoD-Cre), endothelium and endothelial precursors (Tie2-Cre), and vascular smooth muscle (Smooth Muscle Myosin Heavy Chain-Cre [SMMHC-Cre]). RESULTS: Vascular smooth muscle cells did not contribute to any stage of heterotopic ossification in either mouse model. Despite the osteogenic response of cultured skeletal myoblasts to BMPs, skeletal muscle precursors in vivo contributed minimally to heterotopic ossification (<5%), and this contribution was not increased by cardiotoxin injection, which induces muscle regeneration and mobilizes muscle stem cells. In contrast, cells that expressed the vascular endothelial marker Tie2/Tek at some time in their developmental history contributed robustly to the fibroproliferative, chondrogenic, and osteogenic stages of the evolving heterotopic endochondral anlagen. Importantly, endothelial markers were expressed by cells at all stages of heterotopic ossification. Finally, muscle injury and associated inflammation were sufficient to trigger fibrodysplasia ossificans progressiva-like heterotopic ossification in a setting of chronically stimulated BMP activity. CONCLUSIONS: Tie2-expressing progenitor cells, which are endothelial precursors, respond to an inflammatory trigger, differentiate through an endochondral pathway, contribute to every stage of the heterotopic endochondral anlagen, and form heterotopic bone in response to overactive BMP signaling in animal models of fibrodysplasia ossificans progressiva. Thus, the ectopic skeleton is not only supplied by a rich vasculature, but appears to be constructed in part by cells of vascular origin. Further, these data strongly suggest that dysregulation of the BMP signaling pathway and an inflammatory microenvironment are both required for the formation of fibrodysplasia ossificans progressiva-like lesions.
Assuntos
Ossificação Heterotópica/fisiopatologia , Receptor TIE-2/metabolismo , Células-Tronco/fisiologia , Animais , Proteína Morfogenética Óssea 2/farmacologia , Linhagem da Célula , Modelos Animais de Doenças , Endotélio Vascular/citologia , Imuno-Histoquímica , Injeções Intramusculares , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Proteína MyoD/metabolismo , Mioblastos/fisiologia , Miosite Ossificante/fisiopatologia , Ossificação Heterotópica/patologia , Receptores Proteína Tirosina Quinases/metabolismo , Células-Tronco/metabolismoRESUMO
Metamorphosis, the transformation of one normal tissue or organ system into another, is a biological process rarely studied in higher vertebrates or mammals, but exemplified pathologically by the extremely disabling autosomal dominant disorder fibrodysplasia ossificans progressiva (FOP). The recurrent single nucleotide missense mutation in the gene encoding activin receptor IA/activin-like kinase-2 (ACVR1/ALK2), a bone morphogenetic protein type I receptor that causes skeletal metamorphosis in all classically affected individuals worldwide, is the first identified human metamorphogene. Physiological studies of this metamorphogene are beginning to provide deep insight into a highly conserved signaling pathway that regulates tissue stability following morphogenesis, and that when damaged at a highly specific locus (c.617G > A; R206H), and triggered by an inflammatory stimulus permits the renegade metamorphosis of normal functioning connective tissue into a highly ramified skeleton of heterotopic bone. A comprehensive understanding of the process of skeletal metamorphosis, as revealed by the rare condition FOP, will lead to the development of more effective treatments for FOP and, possibly, for more common disorders of skeletal metamorphosis.
Assuntos
Osso e Ossos/patologia , Osso e Ossos/fisiologia , Miosite Ossificante/patologia , Miosite Ossificante/fisiopatologia , Esqueleto , Receptores de Ativinas Tipo I/genética , Animais , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/anatomia & histologia , Humanos , Sistema Imunitário/fisiologia , Anormalidades Musculoesqueléticas/patologia , Mutação de Sentido Incorreto , Miosite Ossificante/diagnóstico , Miosite Ossificante/epidemiologia , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Engenharia TecidualRESUMO
Phenotypic heterogeneity has been observed among mesenchymal stem/stromal cell (MSC) populations, but specific genes associated with this variability have not been defined. To study this question, we analyzed two distinct isogenic MSC populations isolated from umbilical cord blood (UCB1 and UCB2). The use of isogenic populations eliminated differences contributed by genetic background. We characterized these UCB MSCs for cell morphology, growth kinetics, immunophenotype, and potential for differentiation. UCB1 displayed faster growth kinetics, higher population doublings, and increased adipogenic lineage differentiation compared to UCB2. However, osteogenic differentiation was stronger for the UCB2 population. To identify MSC-specific genes and developmental genes associated with observed phenotypic differences, we performed expression analysis using Affymetrix microarrays and compared them to bone marrow (BM) MSCs. We compared UCB1, UCB2, and BM and identified distinct gene expression patterns. Selected clusters were analyzed demonstrating that genes of multiple developmental pathways, such as transforming growth factor-beta (TGF-beta) and wnt genes, and markers of early embryonic stages and mesodermal differentiation displayed significant differences among the MSC populations. In undifferentiated UCB1 cells, multiple genes were significantly up-regulated (p < 0.0001): peroxisome proliferation activated receptor gamma (PPARG), which correlated with adipogenic differentiation capacities, hepatocyte growth factor (HGF), and stromal-derived factor 1 (SDF1/CXCL12), which could both potentially contribute to the higher growth kinetics observed in UCB1 cells. Overall, the results confirmed the presence of two distinct isogenic UCB-derived cell populations, identified gene profiles useful to distinguish MSC types with different lineage differentiation potentials, and helped clarify the heterogeneity observed in these cells.