RESUMEN
INTRODUCTION: The filamins are cytoskeletal binding proteins that dynamically crosslink actin into orthogonal networks or bundle it into stress fibres. The domain structure of filamin proteins is very well characterised, with an N-terminal actin-binding region, followed by 24 immunoglobulin-like repeat units. The repeat domains are separated into distinct segments by two regions of low-complexity known as hinge-1 and hinge-2. The role of hinge-1 especially has been proposed to be essential for protein function as it provides flexibility to the otherwise rigid protein, and is a target for cleavage by calpain. Hinge-1 protects cells from otherwise destructive forces, and the products of calpain cleavage are involved in critical cellular signalling processes, such as survival during hypoxia. Pathogenic variants in FLNA encoding Filamin A, including those that remove the hinge-1 domain, cause a wide range of survivable developmental disorders. In contrast, complete loss of function of this gene is embryonic lethal in human and mouse. METHODS AND RESULTS: In this study, we show that removing filamin A hinge-1 from mouse (FlnaΔH1), while preserving its expression level leads to no obvious developmental phenotype. Detailed characterisation of the skeletons of FlnaΔH1 mice showed no skeletal phenotype reminiscent of that found in the FLNA-causing skeletal dysplasia. Furthermore, nuclear functions of FLNA are maintained with loss of Filamin A hinge-1. CONCLUSION: We conclude that hinge-1 is dispensable for filamin A protein function during development over the murine lifespan.
RESUMEN
Frontometaphyseal dysplasia (FMD) is a progressive sclerosing skeletal dysplasia affecting the long bones and skull. The cause of FMD in some individuals is gain-of-function mutations in FLNA, although how these mutations result in a hyperostotic phenotype remains unknown. Approximately one half of individuals with FMD have no identified mutation in FLNA and are phenotypically very similar to individuals with FLNA mutations, except for an increased tendency to form keloid scars. Using whole-exome sequencing and targeted Sanger sequencing in 19 FMD-affected individuals with no identifiable FLNA mutation, we identified mutations in two genes-MAP3K7, encoding transforming growth factor ß (TGF-ß)-activated kinase (TAK1), and TAB2, encoding TAK1-associated binding protein 2 (TAB2). Four mutations were found in MAP3K7, including one highly recurrent (n = 15) de novo mutation (c.1454C>T [ p.Pro485Leu]) proximal to the coiled-coil domain of TAK1 and three missense mutations affecting the kinase domain (c.208G>C [p.Glu70Gln], c.299T>A [p.Val100Glu], and c.502G>C [p.Gly168Arg]). Notably, the subjects with the latter three mutations had a milder FMD phenotype. An additional de novo mutation was found in TAB2 (c.1705G>A, p.Glu569Lys). The recurrent mutation does not destabilize TAK1, or impair its ability to homodimerize or bind TAB2, but it does increase TAK1 autophosphorylation and alter the activity of more than one signaling pathway regulated by the TAK1 kinase complex. These findings show that dysregulation of the TAK1 complex produces a close phenocopy of FMD caused by FLNA mutations. Furthermore, they suggest that the pathogenesis of some of the filaminopathies caused by FLNA mutations might be mediated by misregulation of signaling coordinated through the TAK1 signaling complex.
Asunto(s)
Frente/anomalías , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Mutación/genética , Osteocondrodisplasias/genética , Transducción de Señal/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Femenino , Filaminas/genética , Humanos , Sistema de Señalización de MAP Quinasas/genética , Masculino , FN-kappa B/metabolismo , Osteocondrodisplasias/metabolismo , Fosforilación , Unión Proteica , Multimerización de ProteínaRESUMEN
The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to the understanding of bone development within the epiphyseal growth plate, factors that regulate periosteal osteogenesis have not been studied as intensively. Osteofibrous dysplasia (OFD) is a congenital disorder of osteogenesis and is typically sporadic and characterized by radiolucent lesions affecting the cortical bone immediately under the periosteum of the tibia and fibula. We identified germline mutations in MET, encoding a receptor tyrosine kinase, that segregate with an autosomal-dominant form of OFD in three families and a mutation in a fourth affected subject from a simplex family and with bilateral disease. Mutations identified in all families with dominant inheritance and in the one simplex subject with bilateral disease abolished the splice inclusion of exon 14 in MET transcripts, which resulted in a MET receptor (MET(Δ14)) lacking a cytoplasmic juxtamembrane domain. Splice exclusion of this domain occurs during normal embryonic development, and forced induction of this exon-exclusion event retarded osteoblastic differentiation in vitro and inhibited bone-matrix mineralization. In an additional subject with unilateral OFD, we identified a somatic MET mutation, also affecting exon 14, that substituted a tyrosine residue critical for MET receptor turnover and, as in the case of the MET(Δ14) mutations, had a stabilizing effect on the mature protein. Taken together, these data show that aberrant MET regulation via the juxtamembrane domain subverts core MET receptor functions that regulate osteogenesis within cortical diaphyseal bone.
Asunto(s)
Enfermedades del Desarrollo Óseo/genética , Exones , Mutación de Línea Germinal , Osteogénesis/genética , Periostio/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Adulto , Secuencia de Bases , Enfermedades del Desarrollo Óseo/metabolismo , Enfermedades del Desarrollo Óseo/patología , Diferenciación Celular , Niño , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes Dominantes , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Osteoblastos/metabolismo , Osteoblastos/patología , Linaje , Periostio/crecimiento & desarrollo , Periostio/patología , Cultivo Primario de Células , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-met/metabolismo , Empalme del ARNRESUMEN
Frontometaphyseal dysplasia (FMD) is caused by gain-of-function mutations in the X-linked gene FLNA in approximately 50% of patients. Recently we characterized an autosomal dominant form of FMD (AD-FMD) caused by mutations in MAP3K7, which accounts for the condition in the majority of patients who lack a FLNA mutation. We previously also described a patient with a de novo variant in TAB2, which we hypothesized was causative of another form of AD-FMD. In this study, a cohort of 20 individuals with AD-FMD is clinically evaluated. This cohort consists of 15 individuals with the recently described, recurrent mutation (c.1454C>T) in MAP3K7, as well as three individuals with missense mutations that result in substitutions in the N-terminal kinase domain of TGFß-activated kinase 1 (TAK1), encoded by MAP3K7. Additionally, two individuals have missense variants in the gene TAB2, which encodes a protein with a close functional relationship to TAK1, TAK1-associated binding protein 2 (TAB2). Although the X-linked and autosomal dominant forms of FMD are very similar, there are distinctions to be made between the two conditions. Individuals with AD-FMD have characteristic facial features, and are more likely to be deaf, have scoliosis and cervical fusions, and have a cleft palate. Furthermore, there are features only found in AD-FMD in our review of the literature including valgus deformity of the feet and predisposition to keloid scarring. Finally, intellectual disability is present in a small number of subjects with AD-FMD but has not been described in association with X-linked FMD.
RESUMEN
Excess exogenous retinoic acid (RA) has been well documented to have teratogenic effects in the limb and craniofacial skeleton. Malformations that have been observed in this context include craniosynostosis, a common developmental defect of the skull that occurs in 1 in 2500 individuals and results from premature fusion of the cranial sutures. Despite these observations, a physiological role for RA during suture formation has not been demonstrated. Here, we present evidence that genetically based alterations in RA signaling interfere with human development. We have identified human null and hypomorphic mutations in the gene encoding the RA-degrading enzyme CYP26B1 that lead to skeletal and craniofacial anomalies, including fusions of long bones, calvarial bone hypoplasia, and craniosynostosis. Analyses of murine embryos exposed to a chemical inhibitor of Cyp26 enzymes and zebrafish lines with mutations in cyp26b1 suggest that the endochondral bone fusions are due to unrestricted chondrogenesis at the presumptive sites of joint formation within cartilaginous templates, whereas craniosynostosis is induced by a defect in osteoblastic differentiation. Ultrastructural analysis, in situ expression studies, and in vitro quantitative RT-PCR experiments of cellular markers of osseous differentiation indicate that the most likely cause for these phenomena is aberrant osteoblast-osteocyte transitioning. This work reveals a physiological role for RA in partitioning skeletal elements and in the maintenance of cranial suture patency.
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Suturas Craneales , Craneosinostosis , Sistema Enzimático del Citocromo P-450 , Tretinoina , Proteínas de Pez Cebra/genética , Animales , Diferenciación Celular , Suturas Craneales/efectos de los fármacos , Suturas Craneales/embriología , Suturas Craneales/crecimiento & desarrollo , Suturas Craneales/patología , Craneosinostosis/enzimología , Craneosinostosis/genética , Craneosinostosis/patología , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/genética , Modelos Animales de Enfermedad , Femenino , Muerte Fetal/genética , Regulación del Desarrollo de la Expresión Génica , Crecimiento y Desarrollo/genética , Humanos , Ratones , Osteoblastos/citología , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Polimorfismo Genético/genética , Embarazo , Ácido Retinoico 4-Hidroxilasa , Homología de Secuencia de Aminoácido , Tretinoina/metabolismo , Tretinoina/farmacología , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
Mutations in FLNA, which encodes the cytoskeletal protein FLNA, cause a spectrum of sclerosing skeletal dysplasias. Although many of these genetic variants are recurrent and cluster within the gene, the pathogenic mechanism that underpins the development of these skeletal phenotypes is unknown. To determine if the skeletal dysplasia in FLNA-related conditions is due to a cell-autonomous loss-of-function localising to osteoblasts and/or osteocytes, we utilised mouse models to conditionally remove Flna from this cellular lineage. Flna was conditionally knocked out from mature osteocytes using the Dmp1-promoter driven Cre-recombinase expressing mouse, as well as the committed osteoblast lineage using the Osx-Cre or Col1a1-Cre expressing lines. We measured skeletal parameters with µCT and histological methods, as well as gene expression in the mineralised skeleton. We found no measureable differences between the conditional Flna knockout mice, and their control littermate counterparts. Moreover, all of the conditional Flna knockout mice, developed and aged normally. From this we concluded that the skeletal dysplasia phenotype associated with pathogenic variants in FLNA is not caused by a cell-autonomous loss-of-function in the osteoblast-osteocyte lineage, adding more evidence to the hypothesis that these phenotypes are due to gain-of-function in FLNA.
RESUMEN
Dominant missense mutations in FLNB, encoding the actin-cross linking protein filamin B (FLNB), cause a broad range of skeletal dysplasias with varying severity by an unknown mechanism. Here these FLNB mutations are shown to cluster in exons encoding the actin-binding domain (ABD) and filamin repeats surrounding the flexible hinge 1 region of the FLNB rod domain. Despite being positioned in domains that bind actin, it is unknown if these mutations perturb cytoskeletal structure. Expression of several full-length FLNB constructs containing ABD mutations resulted in the appearance of actin-containing cytoplasmic focal accumulations of the substituted protein to a degree that was correlated with the severity of the associated phenotypes. In contrast, study of mutations leading to substitutions in the FLNB rod domain that result in the same phenotypes as ABD mutations demonstrated that with only one exception disease-associated substitutions, surrounding hinge 1 demonstrated no tendency to form actin-filamin foci. The exception, a substitution in filamin repeat 6, lies within a region previously implicated in filamin-actin binding. These data are consistent with mutations in the ABD conferring enhanced actin-binding activity but suggest that substitutions affecting repeats near the flexible hinge region of FLNB precipitate the same phenotypes through a different mechanism.
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Actinas/metabolismo , Proteínas Contráctiles/genética , Proteínas Contráctiles/metabolismo , Citoplasma/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Anomalías Musculoesqueléticas/genética , Mutación , Osteocondrodisplasias/genética , Sitios de Unión , Enanismo/genética , Facies , Filaminas , Humanos , Secuencias Repetitivas de Ácidos Nucleicos , Índice de Severidad de la EnfermedadRESUMEN
To assess the effect of Loeys-Dietz syndrome (LDS) mutations affecting TGFΒR1 a selection of seven disease-associated amino acid substitutions were introduced into wild type TGFßR1 and constitutively active TGFßR1(T204D). Receptor function was tested by co-transfection with a luciferase reporter or EGFP-tagged SMAD2 in HEK293 cells. All of the mutations were found to be inactivating for canonical TGF-ß signaling. Differences in residual activity were not found to correlate with disease subtype. In co-transfection experiments with equal amounts wild-type receptor, the LDS mutations were found to confer a modest dominant negative effect. These results are discussed in relation to LDS and the related Marfan syndrome.
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Mutación , Proteínas Serina-Treonina Quinasas/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Aneurisma de la Aorta/genética , Genes Dominantes , Células HEK293 , Humanos , Síndrome de Loeys-Dietz/genética , Síndrome de Marfan/genética , Fenotipo , Fosforilación , Receptor Tipo I de Factor de Crecimiento Transformador beta , Transducción de Señal/genética , Proteínas Smad/metabolismo , Transfección , Factor de Crecimiento Transformador beta/genéticaRESUMEN
CONTEXT: The WNT/ß-catenin pathway is central to the pathogenesis of various human diseases including those affecting bone development and tumor progression. OBJECTIVE: To evaluate the role of a gain-of-function variant in CTNNB1 in a child with a sclerosing bone dysplasia and an adrenocortical adenoma. DESIGN: Whole exome sequencing with corroborative biochemical analyses. PATIENTS: We recruited a child with a sclerosing bone dysplasia and an adrenocortical adenoma together with her unaffected parents. INTERVENTION: Whole exome sequencing and performance of immunoblotting and luciferase-based assays to assess the cellular consequences of a de novo variant in CTNNB1. MAIN OUTCOME MEASURE(S)/RESULT: A de novo variant in CTNNB1 (c.131C>T; p.[Pro44Leu]) was identified in a patient with a sclerosing bone dysplasia and an adrenocortical adenoma. A luciferase-based transcriptional assay of WNT signaling activity verified that the activity of ß-catenin was increased in the cells transfected with a CTNNB1p.Pro44Leu construct (Pâ =â 4.00â ×â 10-5). The ß-catenin p.Pro44Leu variant was also associated with a decrease in phosphorylation at Ser45 and Ser33/Ser37/Thr41 in comparison to a wild-type (WT) CTNNB1 construct (Pâ =â 2.16â ×â 10-3, Pâ =â 9.34â ×â 10-8 respectively). CONCLUSION: Increased ß-catenin activity associated with a de novo gain-of-function CTNNB1 variant is associated with osteosclerotic phenotype and adrenocortical neoplasia.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/patología , Carcinoma Corticosuprarrenal/patología , Enfermedades del Desarrollo Óseo/patología , Mutación , beta Catenina/genética , Neoplasias de la Corteza Suprarrenal/genética , Carcinoma Corticosuprarrenal/genética , Enfermedades del Desarrollo Óseo/genética , Femenino , Humanos , Recién Nacido , Masculino , Linaje , Fenotipo , Pronóstico , Secuenciación del ExomaRESUMEN
Glutamate is the main excitatory neurotransmitter of the CNS. Tissue-type plasminogen activator (tPA) is recognized as a modulator of glutamatergic neurotransmission. This attribute is exemplified by its ability to potentiate calcium signaling following activation of the glutamate-binding NMDA receptor (NMDAR). It has been hypothesized that tPA can directly cleave the NR1 subunit of the NMDAR and thereby potentiate NMDA-induced calcium influx. In contrast, here we show that this increase in NMDAR signaling requires tPA to be proteolytically active, but does not involve cleavage of the NR1 subunit or plasminogen. Rather, we demonstrate that enhancement of NMDAR function by tPA is mediated by a member of the low-density lipoprotein receptor (LDLR) family. Hence, this study proposes a novel functional relationship between tPA, the NMDAR, a LDLR and an unknown substrate which we suspect to be a serpin. Interestingly, whilst tPA alone failed to cleave NR1, cell-surface NMDARs did serve as an efficient and discrete proteolytic target for plasmin. Hence, plasmin and tPA can affect the NMDAR via distinct avenues. Altogether, we find that plasmin directly proteolyses the NMDAR whilst tPA functions as an indirect modulator of NMDA-induced events via LDLR engagement.
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Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/fisiología , Activador de Tejido Plasminógeno/farmacología , Factores de Edad , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Animales Recién Nacidos , Calcio/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Interacciones Farmacológicas , Fibrinolisina/farmacología , Ácido Glutámico/farmacología , Glicina/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Oocitos , Técnicas de Placa-Clamp , Nexinas de Proteasas , Ratas , Receptores de Superficie Celular/metabolismo , Trombina/farmacología , Xenopus laevis , Proteínas de Unión al GTP rap/farmacologíaRESUMEN
Tissue-type plasminogen activator (t-PA) has recently been identified as a modulator of neuronal plasticity and can initiate conversion of the pro-form of brain-derived neurotrophic factor (BDNF) into its mature form. BDNF also increases t-PA gene expression implicating t-PA as a downstream effector of BDNF function. Here we demonstrate that BDNF-mediated induction of t-PA mRNA requires an increase in t-PA gene transcription. Reporter constructs harboring 9.5 kb of the human t-PA promoter conferred BDNF-responsiveness in transfected mouse primary cortical neurons. This regulation was recapitulated in HEK 293 cells coexpressing the TrkB neurotrophin receptor. t-PA promoter-deletion analysis revealed the presence of two BDNF-responsive domains, one located between -3.07 and -2.5 kb and the other within the proximal promoter. The upstream region was shown to confer BDNF responsiveness in a TrkB-dependent manner when attached to a heterologous promoter. We also identify homologous regions within the murine and bovine t-PA gene promoters and demonstrate that the equivalent upstream murine sequence functions as a BDNF-responsive enhancer when inserted 5' of the human proximal t-PA promoter. Hence, BDNF-mediated induction of t-PA transcription relies on conserved modular promoter elements including a novel upstream BDNF-responsive domain and the proximal t-PA gene promoter.
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Factor Neurotrófico Derivado del Encéfalo/química , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Secuencia Conservada , Regulación Enzimológica de la Expresión Génica/fisiología , Regiones Promotoras Genéticas , Activador de Tejido Plasminógeno/química , Activador de Tejido Plasminógeno/genética , Animales , Secuencia de Bases , Factor Neurotrófico Derivado del Encéfalo/fisiología , Bovinos , Línea Celular , Línea Celular Tumoral , Elementos de Facilitación Genéticos/fisiología , Genes Reporteros , Humanos , Ratones , Datos de Secuencia Molecular , Neuronas/enzimología , Neuronas/metabolismo , Activador de Tejido Plasminógeno/fisiologíaRESUMEN
Oncostatin M (OsM) is a member of the interleukin (IL)-6 family of cytokines and is well known for its role in inflammation, cell proliferation, and hematopoiesis. OsM, together with its glycoprotein 130 containing receptor complex, is expressed and regulated in most cells of the central nervous system (CNS), yet the function of OsM within this compartment is poorly understood. Here we have investigated the effect of OsM using in vitro and in vivo models of excitotoxic injury. Using primary cultures of mouse cortical neurons, OsM was shown to reduce N-methyl-D-aspartate (NMDA) -induced neuronal death by 50% when added simultaneously with NMDA while pretreatment of neurons with OsM fully prevented NMDA toxicity indicating a profound protective effect of this cytokine. OsM was also shown to inhibit NMDA-mediated increase in levels of free intracellular calcium and to selectively reduce neuronal expression of the NR2C subunit of the NMDA receptor. Finally, using an in vivo model of excitotoxic injury, OsM significantly reduced the NMDA-induced lesion volume when coinjected with NMDA into the mouse striatum. Taken together, these results identify OsM as a powerful neuroprotective cytokine and provide a rational foundation to explore the therapeutic potential for OsM in diseases of the CNS.
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Lesiones Encefálicas/prevención & control , Neuronas/fisiología , Fármacos Neuroprotectores/farmacología , Oncostatina M/uso terapéutico , Animales , Corteza Cerebral/fisiopatología , Humanos , Mediadores de Inflamación/uso terapéutico , Ratones , N-Metilaspartato/farmacología , Receptores de Oncostatina M/fisiologíaRESUMEN
The melanocortin 4 receptor (MC4R) plays a critical role in the regulation of energy homeostasis, and the MC4R knockout mouse and humans with MC4R defective mutations in only one allele indicate that there is a gene dosage effect. Alterations in gene expression levels for MC4R could, therefore, have significant effects on energy homeostasis. To begin to develop a mouse model for studies on MC4R promoter in situ we used approximately 1 kb mouse MC4R promoter together with 426 bp MC4R 5' UTR, previously shown to support basal expression of reporter gene transcription in cell lines with endogenous MC4R mRNA, and fused this DNA to a nuclear localized LacZ reporter gene. The construct was injected into pronuclei from FVB mice. Five transgenic lines were identified as carrying autosomal transgene insertions; three of these had significant beta-galactosidase staining in brain and in a few cells in the heart but not in kidney, liver, lung, gonadal fat or testis. The pattern of transgene expression in the brain differed markedly for the three lines, and in one of these lines was remarkably similar to endogenous MC4R mRNA expression observed using in situ hybridisation. In conclusion, approximately 1 kb mouse MC4R promoter is sufficient to direct gene expression to the brain including regions that express endogenous MC4R mRNA.