RESUMEN
Cysteine-rich transmembrane bone morphogenetic protein regulator 1 (CRIM1) is a type I transmembrane protein involved in the organogenesis of many tissues via its interactions with growth factors including BMP, TGF-ß, and VEGF. In this study, we used whole-exome sequencing and linkage analysis to identify a novel Crim1 mutant allele generated by ENU mutagenesis in mice. This allele is a missense mutation that causes a cysteine-to-serine substitution at position 140, and is referred to as Crim1C140S. In addition to the previously reported phenotypes in Crim1 mutants, Crim1C140S homozygous mice exhibited several novel phenotypes, including dwarfism, enlarged seminal vesicles, and rectal prolapse. In vitro analyses showed that Crim1C140S mutation affected the formation of CRIM1 complexes and decreased the amount of the overexpressed CRIM1 proteins in the cell culture supernatants. Cys140 is located in the internal region 1 (IR1) of the N-terminal extracellular region of CRIM1 and resides outside any identified functional domains. Inference of the domain architecture suggested that the Crim1C140S mutation disturbs an intramolecular disulfide bond in IR1, leading to the protein instability and the functional defects of CRIM1. Crim1C140S highlights the functional importance of the IR1, and Crim1C140S mice should serve as a valuable model for investigating the functions of CRIM1 that are unidentified as yet.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas/química , Receptores de Proteínas Morfogenéticas Óseas/genética , Cisteína/química , Alelos , Secuencia de Aminoácidos , Animales , Ratones , Ratones Mutantes , Mutación/genética , Fenotipo , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
In humans, mutations in the COL2A1 gene encoding the α1(II) chain of type II collagen, create many clinical phenotypes collectively termed type II collagenopathies. However, the mechanisms generating this diversity remain to be determined. Here we identified a novel Col2a1 mutant mouse line by screening a large-scale N-ethyl-N-nitrosourea mutant mouse library. This mutant possessed a p.Tyr1391Ser missense mutation in the C-propeptide coding region, and this mutation was located in positions corresponding to the human COL2A1 mutation responsible for platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T). As expected, p.Tyr1391Ser homozygotes exhibited lethal skeletal dysplasias resembling PLSD-T, including extremely short limbs and severe dysplasia of the spine and pelvis. The secretion of the mutant proteins into the extracellular space was disrupted, accompanied by an abnormally expanded endoplasmic reticulum (ER) and the up-regulation of ER stress-related genes in chondrocytes. Chondrocyte apoptosis was severely induced in the growth plate of the homozygotes. These findings strongly suggest that ER stress-mediated apoptosis caused by the accumulated mutant proteins in ER contributes to skeletal dysplasia in Co12a1 mutant mice and PLSD-T patients.
Asunto(s)
Apoptosis , Colágeno Tipo II/genética , Estrés del Retículo Endoplásmico , Displasia Tanatofórica/genética , Animales , Condrocitos/metabolismo , Condrocitos/patología , Femenino , Placa de Crecimiento/anomalías , Placa de Crecimiento/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación Missense , Esqueleto/anomalías , Displasia Tanatofórica/patología , Respuesta de Proteína DesplegadaRESUMEN
Microphthalmia with limb anomalies (MLA) is a rare autosomal-recessive disorder, presenting with anophthalmia or microphthalmia and hand and/or foot malformation. We mapped the MLA locus to 14q24 and successfully identified three homozygous (one nonsense and two splice site) mutations in the SPARC (secreted protein acidic and rich in cysteine)-related modular calcium binding 1 (SMOC1) in three families. Smoc1 is expressed in the developing optic stalk, ventral optic cup, and limbs of mouse embryos. Smoc1 null mice recapitulated MLA phenotypes, including aplasia or hypoplasia of optic nerves, hypoplastic fibula and bowed tibia, and syndactyly in limbs. A thinned and irregular ganglion cell layer and atrophy of the anteroventral part of the retina were also observed. Soft tissue syndactyly, resulting from inhibited apoptosis, was related to disturbed expression of genes involved in BMP signaling in the interdigital mesenchyme. Our findings indicate that SMOC1/Smoc1 is essential for ocular and limb development in both humans and mice.
Asunto(s)
Deformidades Congénitas de las Extremidades/genética , Microftalmía/genética , Osteonectina/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 14/genética , Codón sin Sentido/genética , Extremidades/crecimiento & desarrollo , Ojo/crecimiento & desarrollo , Genes Recesivos , Sitios Genéticos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Nervio Óptico/anomalías , Empalme del ARN/genética , Síndrome de Waardenburg/genéticaRESUMEN
Proteoglycans are a family of extracellular macromolecules comprised of glycosaminoglycan chains of a repeated disaccharide linked to a central core protein. Proteoglycans have critical roles in chondrogenesis and skeletal development. The glycosaminoglycan chains found in cartilage proteoglycans are primarily composed of chondroitin sulfate. The integrity of chondroitin sulfate chains is important to cartilage proteoglycan function; however, chondroitin sulfate metabolism in mammals remains poorly understood. The solute carrier-35 D1 (SLC35D1) gene (SLC35D1) encodes an endoplasmic reticulum nucleotide-sugar transporter (NST) that might transport substrates needed for chondroitin sulfate biosynthesis. Here we created Slc35d1-deficient mice that develop a lethal form of skeletal dysplasia with severe shortening of limbs and facial structures. Epiphyseal cartilage in homozygous mutant mice showed a decreased proliferating zone with round chondrocytes, scarce matrices and reduced proteoglycan aggregates. These mice had short, sparse chondroitin sulfate chains caused by a defect in chondroitin sulfate biosynthesis. We also identified that loss-of-function mutations in human SLC35D1 cause Schneckenbecken dysplasia, a severe skeletal dysplasia. Our findings highlight the crucial role of NSTs in proteoglycan function and cartilage metabolism, thus revealing a new paradigm for skeletal disease and glycobiology.
Asunto(s)
Huesos/embriología , Cartílago/embriología , Sulfatos de Condroitina/biosíntesis , Proteínas de Transporte de Monosacáridos/fisiología , Proteínas de Transporte de Nucleótidos/fisiología , Animales , Huesos/metabolismo , Huesos/patología , Cartílago/metabolismo , Cartílago/patología , Células Cultivadas , Condrocitos/metabolismo , Condrocitos/patología , Epífisis/embriología , Epífisis/metabolismo , Epífisis/patología , Huesos Faciales/anomalías , Huesos Faciales/embriología , Huesos Faciales/metabolismo , Humanos , Deformidades Congénitas de las Extremidades/embriología , Deformidades Congénitas de las Extremidades/genética , Deformidades Congénitas de las Extremidades/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas de Transporte de Monosacáridos/deficiencia , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Nucleótidos/genéticaRESUMEN
Lung morphogenesis is a well orchestrated, tightly regulated process through several molecular pathways, including TGF-ß/bone morphogenetic protein (BMP) signaling. Alteration of these signaling pathways leads to lung malformation. We investigated the role of Follistatin-like 1 (Fstl1), a secreted follistatin-module-containing glycoprotein, in lung development. Deletion of Fstl1 in mice led to postnatal lethality as a result of respiratory failure. Analysis of the mutant phenotype showed that Fstl1 is essential for tracheal cartilage formation and alveolar maturation. Deletion of the Fstl1 gene resulted in malformed tracheal rings manifested as discontinued rings and reduced ring number. Fstl1-deficient mice displayed septal hypercellularity and end-expiratory atelectasis, which were associated with impaired differentiation of distal alveolar epithelial cells and insufficient production of mature surfactant proteins. Mechanistically, Fstl1 interacted directly with BMP4, negatively regulated BMP4/Smad1/5/8 signaling, and inhibited BMP4-induced surfactant gene expression. Reducing BMP signaling activity by Noggin rescued pulmonary atelectasis of Fstl1-deficient mice. Therefore, we provide in vivo and in vitro evidence to demonstrate that Fstl1 modulates lung development and alveolar maturation, in part, through BMP4 signaling.
Asunto(s)
Proteína Morfogenética Ósea 4/antagonistas & inhibidores , Proteína Morfogenética Ósea 4/metabolismo , Proteínas Relacionadas con la Folistatina/metabolismo , Alveolos Pulmonares/embriología , Transducción de Señal/fisiología , Animales , Proteína Morfogenética Ósea 4/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cartílago/citología , Cartílago/embriología , Línea Celular Tumoral , Proteínas Relacionadas con la Folistatina/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Noqueados , Alveolos Pulmonares/citología , Surfactantes Pulmonares/metabolismo , Proteínas Smad/genética , Proteínas Smad/metabolismo , Tráquea/citología , Tráquea/embriologíaRESUMEN
Disturbed zinc (Zn) homeostasis in mammals is mainly characterized by impaired bone generation accompanied with growth retardation. However, the underlying mechanisms that determine how Zn controls bone homeostasis remain to be defined. Zn homeostasis is tightly controlled by Zn transporter families. Recent studies have shown that Zn transporter-mediated Zn ion (Zn(2+)) behaves as a signaling factor, called Zn signal, that exerts a multiple function in cellular events, showing why Zn has a vital effect on mammalian bone growth and regeneration. This perspective put importance on the principal mechanisms of Zn participation in mammalian bone homeostasis, shifting our focus on the role of Zn from simply a nutrient to a signaling molecule that fine-tunes intracellular signaling events.
Asunto(s)
Huesos/metabolismo , Transducción de Señal , Zinc/metabolismo , Animales , Condrocitos/citología , Condrocitos/metabolismo , Humanos , Osteoblastos/citología , Osteoblastos/metabolismo , OsteogénesisRESUMEN
Core-binding factor beta (CBFbeta, also called polyomavirus enhancer binding protein 2beta (PEBP2B)) is associated with an inversion of chromosome 16 and is associated with acute myeloid leukemia in humans. CBFbeta forms a heterodimer with RUNX1 (runt-related transcription factor 1), which has a DNA binding domain homologous to the pair-rule protein runt in Drosophila melanogaster. Both RUNX1 and CBFbeta are essential for hematopoiesis. Haploinsufficiency of another runt-related protein, RUNX2 (also called CBFA1), causes cleidocranial dysplasia in humans and is essential in skeletal development by regulating osteoblast differentiation and chondrocyte maturation. Mice deficient in Cbfb (Cbfb(-/-)) die at midgestation, so the function of Cbfbeta in skeletal development has yet to be ascertained. To investigate this issue, we rescued hematopoiesis of Cbfb(-/-) mice by introducing Cbfb using the Gata1 promoter. The rescued Cbfb(-/-) mice recapitulated fetal liver hematopoiesis in erythroid and megakaryocytic lineages and survived until birth, but showed severely delayed bone formation. Although mesenchymal cells differentiated into immature osteoblasts, intramembranous bones were poorly formed. The maturation of chondrocytes into hypertrophic cells was markedly delayed, and no endochondral bones were formed. Electrophoretic mobility shift assays and reporter assays showed that Cbfbeta was necessary for the efficient DNA binding of Runx2 and for Runx2-dependent transcriptional activation. These findings indicate that Cbfbeta is required for the function of Runx2 in skeletal development.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas de Neoplasias , Osteogénesis , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Animales , Biomarcadores/análisis , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Subunidad beta del Factor de Unión al Sitio Principal , Factores de Unión al Sitio Principal , Proteínas de Unión al ADN/genética , Dimerización , Embrión de Mamíferos/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Genes Letales , Hematopoyesis/genética , Hematopoyesis/fisiología , Ratones , Ratones Transgénicos , Osteoblastos/citología , Osteoblastos/fisiología , Fenotipo , Estructura Terciaria de Proteína , ARN Mensajero/análisis , Cráneo/citología , Factor de Transcripción AP-2 , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
There are many steps in the post-translational modification of collagen molecules. When abnormality occurs in some step, the unfolded collagen molecules are accumulated in the endoplasmic reticulum (ER) , leading to ER stress. ER stress also occurs downstream of the defective modification of collagen in bone and cartilage. ER stress-induced apoptosis or ER stress response without inducing apoptosis may be associated with the pathogenesis of genetic collagen disorders in bone and cartilage.
Asunto(s)
Enfermedades Óseas Metabólicas/etiología , Enfermedades Óseas Metabólicas/metabolismo , Huesos/metabolismo , Enfermedades de los Cartílagos/etiología , Enfermedades de los Cartílagos/metabolismo , Cartílago/metabolismo , Colágeno/genética , Colágeno/metabolismo , Estrés del Retículo Endoplásmico/genética , Estrés del Retículo Endoplásmico/fisiología , Animales , Apoptosis , Enfermedades Óseas Metabólicas/genética , Enfermedades de los Cartílagos/genética , Retículo Endoplásmico/metabolismo , Humanos , Ratones , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
SLC35A3 is considered an uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter in mammals and regulates the branching of N-glycans. A missense mutation in SLC35A3 causes complex vertebral malformation (CVM) in cattle. However, the biological functions of SLC35A3 have not been fully clarified. To address these issues, we have established Slc35a3-/-mice using CRISPR/Cas9 genome editing system. The generated mutant mice were perinatal lethal and exhibited chondrodysplasia recapitulating CVM-like vertebral anomalies. During embryogenesis, Slc35a3 mRNA was expressed in the presomitic mesoderm of wild-type mice, suggesting that SLC35A3 transports UDP-GlcNAc used for the sugar modification that is essential for somite formation. In the growth plate cartilage of Slc35a3-/-embryos, extracellular space was drastically reduced, and many flat proliferative chondrocytes were reshaped. Proliferation, apoptosis and differentiation were not affected in the chondrocytes of Slc35a3-/-mice, suggesting that the chondrodysplasia phenotypes were mainly caused by the abnormal extracellular matrix quality. Because these histological abnormalities were similar to those observed in several mutant mice accompanying the impaired glycosaminoglycan (GAG) biosynthesis, GAG levels were measured in the spine and limbs of Slc35a3-/-mice using disaccharide composition analysis. Compared with control mice, the amounts of heparan sulfate, keratan sulfate, and chondroitin sulfate/dermatan sulfate, were significantly decreased in Slc35a3-/-mice. These findings suggest that SLC35A3 regulates GAG biosynthesis and the chondrodysplasia phenotypes were partially caused by the decreased GAG synthesis. Hence, Slc35a3-/- mice would be a useful model for investigating the in vivo roles of SLC35A3 and the pathological mechanisms of SLC35A3-associated diseases.
Asunto(s)
Anomalías Musculoesqueléticas , Osteocondrodisplasias , Animales , Bovinos , Ratones , Transporte Biológico , Sulfato de Queratano , Mamíferos , Nucleótidos , Osteocondrodisplasias/genética , Uridina DifosfatoRESUMEN
BACKGROUND: Desbuquois dysplasia (DD) is a recessively inherited condition characterised by short stature, generalised skeletal dysplasia and advanced bone maturation. DD is both clinically and radiographically heterogeneous, and two subtypes have been distinguished based on the presence (type 1) or absence (type 2) of an accessory metacarpal bone. In addition, an apparently distinct variant without additional metacarpal bone but with short metacarpals and long phalanges (Kim variant) has been described recently. Mutations in the gene that encodes for CANT1 (calcium-activated nucleotidase 1) have been identified in a subset of patients with DD type 1. METHODS: A series of 11 subjects with DD from eight families (one type 1, two type 2, five Kim variant) were examined for CANT1 mutations by direct sequencing of all coding exons and their flanking introns. RESULTS: Eight distinct mutations were identified in seven families (one type 1, one type 2 and all 5 Kim variant): three were nonsense and five were missense. All missense mutations occurred at highly conserved amino acids in the nucleotidase conserved regions of CANT1. Measurement of nucleotidase activity in vitro showed that the missense mutations were all associated with loss-of-function. CONCLUSION: The clinical-radiographic spectrum produced by CANT1 mutations must be extended to include DD type 2 and Kim variant. While presence or absence of an additional metacarpal ossification centre has been used to distinguish subtypes of DD, this sign is not a distinctive criterion to predict the molecular basis in DD.
Asunto(s)
Mutación Missense/genética , Nucleotidasas/genética , Secuencia de Aminoácidos , Animales , Células COS , Preescolar , Chlorocebus aethiops , Anomalías Craneofaciales/clasificación , Anomalías Craneofaciales/complicaciones , Anomalías Craneofaciales/diagnóstico por imagen , Anomalías Craneofaciales/genética , Enanismo/clasificación , Enanismo/complicaciones , Enanismo/diagnóstico por imagen , Enanismo/genética , Mano/diagnóstico por imagen , Deformidades Congénitas de la Mano/complicaciones , Deformidades Congénitas de la Mano/diagnóstico por imagen , Humanos , Inestabilidad de la Articulación/clasificación , Inestabilidad de la Articulación/complicaciones , Inestabilidad de la Articulación/diagnóstico por imagen , Inestabilidad de la Articulación/genética , Datos de Secuencia Molecular , Nucleotidasas/química , Osificación Heterotópica/clasificación , Osificación Heterotópica/complicaciones , Osificación Heterotópica/diagnóstico por imagen , Osificación Heterotópica/genética , Polidactilia/clasificación , Polidactilia/complicaciones , Polidactilia/diagnóstico por imagen , Polidactilia/genética , Radiografía , Alineación de SecuenciaRESUMEN
Lumbar-disc herniation (LDH), one of the most common musculoskeletal diseases, has strong genetic determinants. Recently, several genes that encode extracellular matrix (ECM) proteins in the intervertebral disc have been reported to associate with LDH. Thrombospondins (THBSs) 1 and 2 are good candidates for the LDH susceptibility gene: They are intervertebral disc ECM proteins that regulate the effective levels of matrix metalloproteinases (MMPs) 2 and 9, which are key effectors of ECM remodeling. Here, we report that THBS2 is associated with LDH in Japanese populations. An intronic SNP in THBS2 (IVS10-8C --> T; rs9406328) showed significant association (p = 0.0000028) with LDH in two independent Japanese populations. This SNP, located in a polypyrimidine tract upstream of the 3' splice site of intron 10, exerts allelic differences on exon 11 skipping rates in vivo, with the susceptibility allele showing increased skipping. Skipping of exon 11 results in decreased THBS2 interaction with MMP2 and MMP9. Further, a missense SNP in MMP9 (Q279R; rs17576) is also strongly associated with LDH in the Japanese population (p = 0.00049) and shows a combinatorial effect with THBS2 (odds ratio 3.03, 95% confidence interval 1.58-5.77). Thus, a splicing-affecting SNP in THBS2 and a missense SNP in MMP9 are associated with susceptibility to LDH. Our data indicate that regulation of intervertebral disc ECM metabolism by the THBS2-MMP system plays an essential role in the etiology and pathogenesis of LDH.
Asunto(s)
Empalme Alternativo , Desplazamiento del Disco Intervertebral/genética , Vértebras Lumbares , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Trombospondinas/genética , Adulto , Células Cultivadas , Matriz Extracelular/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Humanos , Desplazamiento del Disco Intervertebral/metabolismo , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Unión Proteica , Trombospondinas/metabolismoRESUMEN
The COL2A1 gene encodes the α1(II) chain of the homotrimeric type II collagen, the most abundant protein in cartilage. In humans, COL2A1 mutations create many clinical phenotypes collectively termed type II collagenopathies; however, the genetic basis of the phenotypic diversity is not well elucidated. Therefore, animal models corresponding to multiple type II collagenopathies are required. In this study we identified a novel Col2a1 missense mutation--c.44406A>C (p.D1469A)--produced by large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis in a mouse line. This mutation was located in the C-propeptide coding region of Col2a1 and in the positions corresponding to a human COL2A1 mutation responsible for platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T). The phenotype was inherited as a semidominant trait. The heterozygotes were mildly but significantly smaller than wild-type mice. The homozygotes exhibited lethal skeletal dysplasias, including extremely short limbs, severe spondylar dysplasia, severe pelvic hypoplasia, and brachydactyly. As expected, these skeletal defects in the homozygotes were similar to those in PLSD-T patients. The secretion of the mutant proteins into the extracellular space was disrupted, accompanied by abnormally expanded rough endoplasmic reticulum (ER) and upregulation of ER stress-related genes, such as Grp94 and Chop, in chondrocytes. These findings suggested that the accumulation of mutant type II collagen in the ER and subsequent induction of ER stress are involved, at least in part in the PLSD-T-like phenotypes of the mutants. This mutant should serve as a good model for studying PLSD-T pathogenesis and the mechanisms that create the great diversity of type II collagenopathies.
Asunto(s)
Colágeno Tipo II/genética , Modelos Animales de Enfermedad , Ratones Mutantes/genética , Osteocondrodisplasias/genética , Azul Alcián , Animales , Antraquinonas , Huesos/ultraestructura , Mapeo Cromosómico , Cartilla de ADN/genética , Etilnitrosourea/toxicidad , Genotipo , Inmunohistoquímica , Ratones , Microscopía Electrónica de Transmisión , Mutagénesis , Mutación Missense/efectos de los fármacos , Mutación Missense/genética , Osteocondrodisplasias/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Desbuquois dysplasia (DBQD) is a severe skeletal dysplasia of autosomal recessive inheritance. DBQD is classified into types 1 and 2 based on presence or absence of hand anomalies. In a previous study, we found a CANT1 (for calcium-activated nucleotidase 1) mutation, c.676G>A in five DBQD families. They were all East Asians (Japanese or Korean). The high prevalence of the same mutation among Japanese and Korean suggested that it is a common founder mutation in the two populations. To examine a possible common founder, we examined the region around CANT1 in chromosomes with c.676G>A mutation by genotyping polymorphic markers in the region for the families. We examined their haplotypes using the family data. We identified in all families a common haplotype containing the CANT1 mutation that ranged up to 550 kb. The two unrelated carriers of the mutation in general populations in Korea and Japan could also have the haplotype. We estimated the age of the founder mutation as â¼ 1420 years (95% CI=880-1940 years). The c.676G>A mutation of CANT1 commonly seen in Japanese and Korean DBQD should be derived from a common founder.
Asunto(s)
Enfermedades del Desarrollo Óseo/genética , Efecto Fundador , Mutación/genética , Nucleotidasas/genética , Haplotipos , Humanos , Japón , Corea (Geográfico) , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
CS (chondroitin sulfate) is a glycosaminoglycan species that is widely distributed in the extracellular matrix. To understand the physiological roles of enzymes involved in CS synthesis, we produced CSGalNAcT1 (CS N-acetylgalactosaminyltransferase 1)-null mice. CS production was reduced by approximately half in CSGalNAcT1-null mice, and the amount of short-chain CS was also reduced. Moreover, the cartilage of the null mice was significantly smaller than that of wild-type mice. Additionally, type-II collagen fibres in developing cartilage were abnormally aggregated and disarranged in the homozygous mutant mice. These results suggest that CSGalNAcT1 is required for normal CS production in developing cartilage.
Asunto(s)
Condrogénesis , Sulfatos de Condroitina/biosíntesis , Placa de Crecimiento/enzimología , N-Acetilgalactosaminiltransferasas/metabolismo , Animales , Western Blotting , Línea Celular , Proliferación Celular , Condrocitos/citología , Condrocitos/enzimología , Condrocitos/metabolismo , Colágeno Tipo II/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Placa de Crecimiento/embriología , Placa de Crecimiento/metabolismo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , N-Acetilgalactosaminiltransferasas/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Ehlers-Danlos syndrome (EDS) is a heterogeneous connective tissue disorder involving skin and joint laxity and tissue fragility. A new type of EDS, similar to kyphoscoliosis type but without lysyl hydroxylase deficiency, has been investigated. We have identified a homozygous CHST14 (carbohydrate sulfotransferase 14) mutation in the two familial cases and compound heterozygous mutations in four sporadic cases. CHST14 encodes dermatan 4-O-sulfotransferase 1 (D4ST1), which transfers active sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of the N-acetyl-D-galactosamine (GalNAc) residues of dermatan sulfate (DS). Transfection experiments of mutants and enzyme assays using fibroblast lysates of patients showed the loss of D4ST1 activity. CHST14 mutations altered the glycosaminoglycan (GAG) components in patients' fibroblasts. Interestingly, DS of decorin proteoglycan, a key regulator of collagen fibril assembly, was completely lost and replaced by chondroitin sulfate (CS) in the patients' fibroblasts, leading to decreased flexibility of GAG chains. The loss of the decorin DS proteoglycan due to CHST14 mutations may preclude proper collagen bundle formation or maintenance of collagen bundles while the sizes and shapes of collagen fibrils are unchanged as observed in the patients' dermal tissues. These findings indicate the important role of decorin DS in the extracellular matrix and a novel pathomechanism in EDS.
Asunto(s)
Síndrome de Ehlers-Danlos/clasificación , Síndrome de Ehlers-Danlos/genética , Mutación/genética , Sulfotransferasas/genética , Adulto , Secuencia de Aminoácidos , Niño , Colágeno/metabolismo , Decorina , Dermis/patología , Síndrome de Ehlers-Danlos/enzimología , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Glicosaminoglicanos/metabolismo , Humanos , Masculino , Modelos Biológicos , Datos de Secuencia Molecular , Linaje , Proteoglicanos/metabolismo , Transducción de Señal , Sulfotransferasas/química , Factor de Crecimiento Transformador beta , Carbohidrato SulfotransferasasRESUMEN
Osteoarthritis (OA) is the most common form of arthritis and is characterized by the gradual loss of articular cartilage. Several OA-susceptibility genes have been identified; however, there are few pharmaceutical targets that can be targeted with small-molecule compounds. To investigate whether a susceptibility gene for OA exists among G-protein-coupled receptors (GPCRs), we performed a stepwise association study for 167 single nucleotide polymorphisms (SNPs) in 44 GPCR genes that were present in cartilage. Through the stepwise association study, an SNP located in the promoter region of EDG2 [endothelial differentiation, lysophosphatidic acid (LPA) GPCR, 2] (-2,820G/A; rs10980705) showed significant association with knee OA in two independent populations (pooled P = 2.6 x 10(-5)). Luciferase and electrophoretic mobility shift assays indicate that this SNP exerts an allelic difference on transcriptional activity and DNA binding in synovial cells, with the susceptibility allele showing increased activity and binding. EDG2 encodes an LPA receptor dominantly expressed in the synovium. The LPA receptor increased the expression of inflammatory cytokines and matrix metalloproteases in synovial cells. Our findings suggest that the LPA-EDG2 signal is involved in the pathogenesis of OA via catabolic process.
Asunto(s)
Predisposición Genética a la Enfermedad , Osteoartritis de la Rodilla/genética , Polimorfismo de Nucleótido Simple , Receptores del Ácido Lisofosfatídico/genética , Anciano , Pueblo Asiatico/genética , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Osteoartritis de la Rodilla/metabolismo , Regiones Promotoras Genéticas , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal , Membrana Sinovial/metabolismoRESUMEN
Desbuquois dysplasia (DD) type 1 is a rare skeletal dysplasia characterized by a short stature, round face, progressive scoliosis, and joint laxity. The causative gene has been identified as calcium-activated nucleotidase 1 (CANT1), which encodes a nucleotidase that preferentially hydrolyzes UDP to UMP and phosphate. In this study, we generated Cant1 KO mice using CRISPR/Cas9-mediated genome editing. All F0 mice possessing frameshift deletions at both Cant1 alleles exhibited a dwarf phenotype. Germline transmission of the edited allele was confirmed in an F0 heterozygous mouse, and KO mice were generated by crossing of the heterozygous breeding pairs. Cant1 KO mice exhibited skeletal defects, including short stature, thoracic kyphosis, and delta phalanx, all of which are observed in DD type 1 patients. The glycosaminoglycan (GAG) content and extracellular matrix space were reduced in the growth plate cartilage of mutants, and proliferating chondrocytes lost their typical flat shape and became round. Chondrocyte differentiation, especially terminal differentiation to hypertrophic chondrocytes, was impaired in Cant1 KO mice. These findings indicate that CANT1 is involved in the synthesis of GAG and regulation of chondrocyte differentiation in the cartilage and contribute to a better understanding of the pathogenesis of DD type 1.
Asunto(s)
Ácido Anhídrido Hidrolasas/deficiencia , Anomalías Craneofaciales/genética , Enanismo/genética , Placa de Crecimiento/patología , Inestabilidad de la Articulación/genética , Osificación Heterotópica/genética , Polidactilia/genética , Ácido Anhídrido Hidrolasas/genética , Animales , Diferenciación Celular/genética , Condrocitos/patología , Anomalías Craneofaciales/diagnóstico , Anomalías Craneofaciales/patología , Modelos Animales de Enfermedad , Enanismo/diagnóstico , Enanismo/patología , Femenino , Glicosaminoglicanos/biosíntesis , Placa de Crecimiento/citología , Placa de Crecimiento/crecimiento & desarrollo , Humanos , Inestabilidad de la Articulación/diagnóstico , Inestabilidad de la Articulación/patología , Masculino , Ratones , Ratones Noqueados , Osificación Heterotópica/diagnóstico , Osificación Heterotópica/patología , Polidactilia/diagnóstico , Polidactilia/patología , Esqueleto/diagnóstico por imagen , Esqueleto/crecimiento & desarrolloRESUMEN
Osteoclasts are bone-resorbing cells that play an essential role in maintaining bone homeostasis. Zinc (Zn) has been reported to inhibit osteoclast-mediated bone resorption, but the mechanism of this action has not been clarified. Zn homeostasis is tightly controlled by the coordinated actions of many Zn transporters. The Zn transporter ZIP14/Slc39a14 is involved in various physiological functions; hence, Zip14-knockout (KO) mice exhibit multiple phenotypes. In this study, we thoroughly investigated the bone phenotypes of Zip14-KO mice, demonstrating that the KO mice exhibited osteopenia in both trabecular and cortical bones. In Zip14-KO mice, bone resorption was increased, whereas the bone formation rate was unchanged. Zip14 mRNA was expressed in normal osteoclasts both in vivo and in vitro, but receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis was not impaired in bone marrow-derived macrophages prepared from Zip14-KO mice. These results suggest that ZIP14 regulates bone homeostasis by inhibiting bore resorption and that in Zip14-KO mice, bone resorption is increased due to the elimination of this inhibitory regulation. Further studies are necessary to conclude whether the enhancement of bone resorption in Zip14-KO mice is due to a cell-autonomous or a non-cell-autonomous osteoclast defect.
RESUMEN
Camurati-Engelmann disease (CED) is a rare sclerosing bone disorder in humans with autosomal dominant inheritance. Mutations in the gene (TGFB1) that encodes transforming growth factor-ß1 (TGF-ß1) are causative for CED. TGF-ß1 signaling is enhanced by the CED-causing mutations. In this study, we performed Tgfb1 mutation screening in an ENU-mutagenized mouse genomic DNA library. We identified a missense mutation in which cysteine was substituted by serine at position 225 (p.C225S), that corresponded to the CED-causing mutation (p.C225R). TGF-ß1 mutant protein carrying p.C225S was secreted normally into the extracellular space. Reporter gene assays showed that the p.C225S mutants enhanced TGF-ß signaling at the same level as p.C225R mutants. We generated p.C225S homozygous mice and confirmed that the mature TGF-ß1 levels in the culture supernatants of the calvarial cells from the homozygotes were significantly higher than those from wild-type mice. Although the skull and femur are sclerotic in CED, these phenotypes were not observed in p.C225S homozygous mice. These results suggest that human and mouse bone tissue react differently to TGF-ß1. These findings are useful to pharmacological studies using mouse models in developing drugs that will target TGF-ß signaling.
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Sustitución de Aminoácidos/genética , Síndrome de Camurati-Engelmann/genética , Etilnitrosourea/toxicidad , Estudios de Asociación Genética , Mutación Missense , Factor de Crecimiento Transformador beta1/genética , Animales , Cisteína , Femenino , Biblioteca de Genes , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Terapia Molecular Dirigida , Mutación Missense/efectos de los fármacos , Fenotipo , Serina , Transducción de Señal/genéticaRESUMEN
The Bcl2 family proteins, Bcl2 and BclXL, suppress apoptosis by preventing the release of caspase activators from mitochondria through the inhibition of Bax subfamily proteins. We reported that BCL2 overexpression in osteoblasts using the 2.3 kb Col1a1 promoter increased osteoblast proliferation, failed to reduce osteoblast apoptosis, inhibited osteoblast maturation, and reduced the number of osteocyte processes, leading to massive osteocyte death. We generated BCLXL (BCL2L1) transgenic mice using the same promoter to investigate BCLXL functions in bone development and maintenance. Bone mineral density in the trabecular bone of femurs was increased, whereas that in the cortical bone was similar to that in wild-type mice. Osteocyte process formation was unaffected and bone structures were similar to those in wild-type mice. A micro-CT analysis showed that trabecular bone volume in femurs and vertebrae and the cortical thickness of femurs were increased. A dynamic bone histomorphometric analysis revealed that the mineralizing surface was larger in trabecular bone, and the bone-formation rate was increased in cortical bone. Serum osteocalcin but not TRAP5b was increased, BrdU-positive osteoblastic cell numbers were increased, TUNEL-positive osteoblastic cell numbers were reduced, and osteoblast marker gene expression was enhanced in BCLXL transgenic mice. The three-point bending test indicated that femurs were stronger in BCLXL transgenic mice than in wild-type mice. The frequency of TUNEL-positive primary osteoblasts was lower in BCLXL transgenic mice than in wild-type mice during cultivation, and osteoblast differentiation was enhanced but depended on cell density, indicating that enhanced differentiation was mainly owing to reduced apoptosis. Increased trabecular and cortical bone volumes were maintained during aging in male and female mice. These results indicate that BCLXL overexpression in osteoblasts increased the trabecular and cortical bone volumes with normal structures and maintained them majorly by preventing osteoblast apoptosis, implicating BCLXL as a therapeutic target of osteoporosis. © 2016 American Society for Bone and Mineral Research.