RESUMEN
Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder affecting tissues of mesenchymal origin. Most individuals with HGPS harbor a de novo c.1824C > T (p.G608G) mutation in the gene encoding lamin A (LMNA), which activates a cryptic splice donor site resulting in production of the toxic "progerin" protein. Clinical manifestations include growth deficiency, lipodystrophy, sclerotic dermis, cardiovascular defects, and bone dysplasia. Here we utilized the LmnaG609G knock-in (KI) mouse model of HGPS to further define mechanisms of bone loss associated with normal and premature aging disorders. Newborn skeletal staining of KI mice revealed altered rib cage shape and spinal curvature, and delayed calvarial mineralization with increased craniofacial and mandibular cartilage content. MicroCT analysis and mechanical testing of adult femurs indicated increased fragility associated with reduced bone mass, recapitulating the progressive bone deterioration that occurs in HGPS patients. We investigated mechanisms of bone loss in KI mice at the cellular level in bone cell populations. Formation of wild-type and KI osteoclasts from marrow-derived precursors was inhibited by KI osteoblast-conditioned media in vitro, suggesting a secreted factor(s) responsible for decreased osteoclasts on KI trabecular surfaces in vivo. Cultured KI osteoblasts exhibited abnormal differentiation characterized by reduced deposition and mineralization of extracellular matrix with increased lipid accumulation compared to wild-type, providing a mechanism for altered bone formation. Furthermore, quantitative analyses of KI transcripts confirmed upregulation of adipogenic genes both in vitro and in vivo. Thus, osteoblast phenotypic plasticity, inflammation and altered cellular cross-talk contribute to abnormal bone formation in HGPS mice.
Asunto(s)
Envejecimiento Prematuro , Enfermedades del Desarrollo Óseo , Progeria , Ratones , Animales , Progeria/genética , Progeria/metabolismo , Mutación , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Diferenciación CelularRESUMEN
Hutchinson-Gilford progeria syndrome (HGPS) is a rare accelerated aging disorder most notably characterized by cardiovascular disease and premature death from myocardial infarction or stroke. The majority of cases are caused by a de novo single nucleotide mutation in the LMNA gene that activates a cryptic splice donor site, resulting in production of a toxic form of lamin A with a 50 amino acid internal deletion, termed progerin. We previously reported the generation of a transgenic murine model of progeria carrying a human BAC harboring the common mutation, G608G, which in the single-copy state develops features of HGPS that are limited to the vascular system. Here, we report the phenotype of mice bred to carry two copies of the BAC, which more completely recapitulate the phenotypic features of HGPS in skin, adipose, skeletal, and vascular tissues. We further show that genetic reduction of the mechanistic target of rapamycin (mTOR) significantly extends lifespan in these mice, providing a rationale for pharmacologic inhibition of the mTOR pathway in the treatment of HGPS.
Asunto(s)
Modelos Animales de Enfermedad , Longevidad , Progeria/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Hutchinson-Gilford progeria syndrome (HGPS) is a rare accelerated aging disorder characterized by premature death from myocardial infarction or stroke. It is caused by de novo single-nucleotide mutations in the LMNA gene that activate a cryptic splice donor site, resulting in the production of a toxic form of lamin A, which is termed progerin. Here we present a potential genetic therapeutic strategy that utilizes antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to block pathogenic splicing of mutant transcripts. Of several candidates, PPMO SRP-2001 provided the most significant decrease in progerin transcripts in patient fibroblasts. Intravenous delivery of SRP-2001 to a transgenic mouse model of HGPS produced significant reduction of progerin transcripts in the aorta, a particularly critical target tissue in HGPS. Long-term continuous treatment with SRP-2001 yielded a 61.6% increase in lifespan and rescue of vascular smooth muscle cell loss in large arteries. These results provide a rationale for proceeding to human trials.
Asunto(s)
Oligonucleótidos Antisentido/uso terapéutico , Progeria/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Transgénicos , Morfolinos/químicaRESUMEN
Hutchinson-Gilford progeria syndrome (HGPS or progeria) is typically caused by a dominant-negative Câ¢G-to-Tâ¢A mutation (c.1824 C>T; p.G608G) in LMNA, the gene that encodes nuclear lamin A. This mutation causes RNA mis-splicing that produces progerin, a toxic protein that induces rapid ageing and shortens the lifespan of children with progeria to approximately 14 years1-4. Adenine base editors (ABEs) convert targeted Aâ¢T base pairs to Gâ¢C base pairs with minimal by-products and without requiring double-strand DNA breaks or donor DNA templates5,6. Here we describe the use of an ABE to directly correct the pathogenic HGPS mutation in cultured fibroblasts derived from children with progeria and in a mouse model of HGPS. Lentiviral delivery of the ABE to fibroblasts from children with HGPS resulted in 87-91% correction of the pathogenic allele, mitigation of RNA mis-splicing, reduced levels of progerin and correction of nuclear abnormalities. Unbiased off-target DNA and RNA editing analysis did not detect off-target editing in treated patient-derived fibroblasts. In transgenic mice that are homozygous for the human LMNA c.1824 C>T allele, a single retro-orbital injection of adeno-associated virus 9 (AAV9) encoding the ABE resulted in substantial, durable correction of the pathogenic mutation (around 20-60% across various organs six months after injection), restoration of normal RNA splicing and reduction of progerin protein levels. In vivo base editing rescued the vascular pathology of the mice, preserving vascular smooth muscle cell counts and preventing adventitial fibrosis. A single injection of ABE-expressing AAV9 at postnatal day 14 improved vitality and greatly extended the median lifespan of the mice from 215 to 510 days. These findings demonstrate the potential of in vivo base editing as a possible treatment for HGPS and other genetic diseases by directly correcting their root cause.
Asunto(s)
Adenina/metabolismo , Edición Génica/métodos , Mutación , Progeria/genética , Progeria/terapia , Alelos , Empalme Alternativo , Animales , Aorta/patología , Emparejamiento Base , Niño , ADN/genética , Modelos Animales de Enfermedad , Femenino , Fibroblastos/metabolismo , Humanos , Lamina Tipo A/química , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Longevidad , Masculino , Ratones , Ratones Transgénicos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Progeria/patología , ARN/genéticaRESUMEN
Hutchinson-Gilford progeria syndrome (HGPS) is a uniformly fatal condition that is especially prevalent in skin, cardiovascular, and musculoskeletal systems. A wide gap exists between our knowledge of the disease and a promising treatment or cure. The aim of this study was to first characterize the musculoskeletal phenotype of the homozygous G608G BAC-transgenic progeria mouse model, and to determine the phenotype changes of HGPS mice after a five-arm preclinical trial of different treatment combinations with lonafarnib, pravastatin, and zoledronic acid. Microcomputed tomography and CT-based rigidity analyses were performed to assess cortical and trabecular bone structure, density, and rigidity. Bones were loaded to failure with three-point bending to assess strength. Contrast-enhanced µCT imaging of mouse femurs was performed to measure glycosaminoglycan content, thickness, and volume of the femoral head articular cartilage. Advanced glycation end products were assessed with a fluorometric assay. The changes demonstrated in the cortical bone structure, rigidity, stiffness, and modulus of the HGPS G608G mouse model may increase the risk for bending and deformation, which could result in the skeletal dysplasia characteristic of HGPS. Cartilage abnormalities seen in this HGPS model resemble changes observed in the age-matched WT controls, including early loss of glycosaminoglycans, and decreased cartilage thickness and volume. Such changes might mimic prevalent degenerative joint diseases in the elderly. Lonafarnib monotherapy did not improve bone or cartilage parameters, but treatment combinations with pravastatin and zoledronic acid significantly improved bone structure and mechanical properties and cartilage structural parameters, which ameliorate the musculoskeletal phenotype of the disease.
Asunto(s)
Conservadores de la Densidad Ósea/uso terapéutico , Modelos Animales de Enfermedad , Lamina Tipo A/genética , Progeria , Envejecimiento/efectos de los fármacos , Envejecimiento/patología , Animales , Huesos/efectos de los fármacos , Huesos/patología , Cartílago/efectos de los fármacos , Cartílago/patología , Fémur/efectos de los fármacos , Fémur/patología , Glicosaminoglicanos/análisis , Articulaciones/efectos de los fármacos , Articulaciones/patología , Lamina Tipo A/metabolismo , Ratones , Ratones Transgénicos , Mutación , Osteoartritis/tratamiento farmacológico , Osteoartritis/patología , Fenotipo , Piperidinas/uso terapéutico , Pravastatina/uso terapéutico , Progeria/tratamiento farmacológico , Progeria/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Piridinas/uso terapéutico , Microtomografía por Rayos X , Ácido Zoledrónico/uso terapéuticoRESUMEN
Melorheostosis is a rare sclerosing dysostosis characterized by asymmetric exuberant bone formation. Recently, we reported that somatic mosaicism for MAP2K1-activating mutations causes radiographical "dripping candle wax" melorheostosis. We now report somatic SMAD3 mutations in bone lesions of four unrelated patients with endosteal pattern melorheostosis. In vitro, the SMAD3 mutations stimulated the TGF-ß pathway in osteoblasts, enhanced nuclear translocation and target gene expression, and inhibited proliferation. Osteoblast differentiation and mineralization were stimulated by the SMAD3 mutation, consistent with higher mineralization in affected than in unaffected bone, but differing from MAP2K1 mutation-positive melorheostosis. Conversely, osteoblast differentiation and mineralization were inhibited when osteogenesis of affected osteoblasts was driven in the presence of BMP2. Transcriptome profiling displayed that TGF-ß pathway activation and ossification-related processes were significantly influenced by the SMAD3 mutation. Co-expression clustering illuminated melorheostosis pathophysiology, including alterations in ECM organization, cell growth, and interferon signaling. These data reveal antagonism of TGF-ß/SMAD3 activation by BMP signaling in SMAD3 mutation-positive endosteal melorheostosis, which may guide future therapies.
Asunto(s)
Melorreostosis/genética , Mutación/genética , Transducción de Señal , Proteína smad3/genética , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba/genética , Animales , Proteína Morfogenética Ósea 2/metabolismo , Huesos/patología , Calcificación Fisiológica , Diferenciación Celular , Línea Celular , Núcleo Celular/metabolismo , Proliferación Celular , Matriz Extracelular/metabolismo , Mutación con Ganancia de Función , Regulación de la Expresión Génica , Humanos , MAP Quinasa Quinasa 1/genética , Melorreostosis/diagnóstico por imagen , Melorreostosis/patología , Ratones , Modelos Biológicos , Osteoblastos/metabolismo , Osteogénesis , Transporte de Proteínas , Transcriptoma/genéticaRESUMEN
Null mutations in CRTAP or P3H1, encoding cartilage-associated protein and prolyl 3-hydroxylase 1, cause the severe bone dysplasias, types VII and VIII osteogenesis imperfecta. Lack of either protein prevents formation of the ER prolyl 3-hydroxylation complex, which catalyzes 3Hyp modification of types I and II collagen and also acts as a collagen chaperone. To clarify the role of the A1 3Hyp substrate site in recessive bone dysplasia, we generated knock-in mice with an α1(I)P986A substitution that cannot be 3-hydroxylated. Mutant mice have normal survival, growth, femoral breaking strength and mean bone mineralization. However, the bone collagen HP/LP crosslink ratio is nearly doubled in mutant mice, while collagen fibril diameter and bone yield energy are decreased. Thus, 3-hydroxylation of the A1 site α1(I)P986 affects collagen crosslinking and structural organization, but its absence does not directly cause recessive bone dysplasia. Our study suggests that the functions of the modification complex as a collagen chaperone are thus distinct from its role as prolyl 3-hydroxylase.
Asunto(s)
Sustitución de Aminoácidos , Colágeno Tipo I/genética , Osteoblastos/citología , Osteogénesis Imperfecta/genética , Animales , Células Cultivadas , Cadena alfa 1 del Colágeno Tipo I , Modelos Animales de Enfermedad , Fibroblastos/citología , Fibroblastos/metabolismo , Técnicas de Sustitución del Gen , Humanos , Hidroxilación , Masculino , Ratones , Osteoblastos/metabolismo , Osteogénesis Imperfecta/metabolismo , FenotipoRESUMEN
Covalent intermolecular cross-linking of collagen is essential for tissue stability. Recent studies have demonstrated that cyclophilin B (CypB), an endoplasmic reticulum (ER)-resident peptidyl-prolyl cis-trans isomerase, modulates lysine (Lys) hydroxylation of type I collagen impacting cross-linking chemistry. However, the extent of modulation, the molecular mechanism and the functional outcome in tissues are not well understood. Here, we report that, in CypB null (KO) mouse skin, two unusual collagen cross-links lacking Lys hydroxylation are formed while neither was detected in wild type (WT) or heterozygous (Het) mice. Mass spectrometric analysis of type I collagen showed that none of the telopeptidyl Lys was hydroxylated in KO or WT/Het mice. Hydroxylation of the helical cross-linking Lys residues was almost complete in WT/Het but was markedly diminished in KO. Lys hydroxylation at other sites was also lower in KO but to a lesser extent. A key glycosylation site, α1(I) Lys-87, was underglycosylated while other sites were mostly overglycosylated in KO. Despite these findings, lysyl hydroxylases and glycosyltransferase 25 domain 1 levels were significantly higher in KO than WT/Het. However, the components of ER chaperone complex that positively or negatively regulates lysyl hydroxylase activities were severely reduced or slightly increased, respectively, in KO. The atomic force microscopy-based nanoindentation modulus were significantly lower in KO skin than WT. These data demonstrate that CypB deficiency profoundly affects Lys post-translational modifications of collagen likely by modulating LH chaperone complexes. Together, our study underscores the critical role of CypB in Lys modifications of collagen, cross-linking and mechanical properties of skin.
Asunto(s)
Ciclofilinas/química , Lisina/química , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/química , Piel/enzimología , Animales , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/genética , Ciclofilinas/genética , Ciclofilinas/ultraestructura , Retículo Endoplásmico/química , Retículo Endoplásmico/enzimología , Glicosilación , Heterocigoto , Hidroxilación , Lisina/genética , Espectrometría de Masas , Ratones , Ratones Noqueados , Microscopía de Fuerza Atómica , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procesamiento Proteico-Postraduccional/genética , Piel/químicaRESUMEN
Mutations in the type I procollagen C-propeptide occur in ~6.5% of Osteogenesis Imperfecta (OI) patients. They are of special interest because this region of procollagen is involved in α chain selection and folding, but is processed prior to fibril assembly and is absent in mature collagen fibrils in tissue. We investigated the consequences of seven COL1A1 C-propeptide mutations for collagen biochemistry in comparison to three probands with classical glycine substitutions in the collagen helix near the C-propeptide and a normal control. Procollagens with C-propeptide defects showed the expected delayed chain incorporation, slow folding and overmodification. Immunofluorescence microscopy indicated that procollagen with C-propeptide defects was mislocalized to the ER lumen, in contrast to the ER membrane localization of normal procollagen and procollagen with helical substitutions. Notably, pericellular processing of procollagen with C-propeptide mutations was defective, with accumulation of pC-collagen and/or reduced production of mature collagen. In vitro cleavage assays with BMP-1⯱â¯PCPE-1 confirmed impaired C-propeptide processing of procollagens containing mutant proα1(I) chains. Overmodified collagens were incorporated into the matrix in culture. Dermal fibrils showed alterations in average diameter and diameter variability and bone fibrils were disorganized. Altered ER-localization and reduced pericellular processing of defective C-propeptides are expected to contribute to abnormal osteoblast differentiation and matrix function, respectively.
Asunto(s)
Colágeno Tipo I/genética , Retículo Endoplásmico/metabolismo , Procolágeno/metabolismo , Rastreo Diferencial de Calorimetría , Células Cultivadas , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Microscopía Fluorescente , Mutación Missense , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/patología , Estructura Terciaria de ProteínaRESUMEN
Melorheostosis is a rare non-hereditary condition characterized by dense hyperostotic lesions with radiographic "dripping candle wax" appearance. Somatic activating mutations in MAP2K1 have recently been identified as a cause of melorheostosis. However, little is known about the development, composition, structure, and mechanical properties of the bone lesions. We performed a multi-method phenotype characterization of material properties in affected and unaffected bone biopsy samples from six melorheostosis patients with MAP2K1 mutations. On standard histology, lesions show a zone with intensively remodeled osteonal-like structure and prominent osteoid accumulation, covered by a shell formed through bone apposition, consisting of compact multi-layered lamellae oriented parallel to the periosteal surface and devoid of osteoid. Compared with unaffected bone, melorheostotic bone has lower average mineralization density measured by quantitative backscattered electron imaging (CaMean: -4.5%, p = 0.04). The lamellar portion of the lesion is even less mineralized, possibly because the newly deposited material has younger tissue age. Affected bone has higher porosity by micro-CT, due to increased tissue vascularity and elevated 2D-microporosity (osteocyte lacunar porosity: +39%, p = 0.01) determined on quantitative backscattered electron images. Furthermore, nano-indentation modulus characterizing material hardness and stiffness was strictly dependent on tissue mineralization (correlation with typical calcium concentration, CaPeak: r = 0.8984, p = 0.0150, and r = 0.9788, p = 0.0007, respectively) in both affected and unaffected bone, indicating that the surgical hardness of melorheostotic lesions results from their lamellar structure. The results suggest a model for pathophysiology of melorheostosis caused by somatic activating mutations in MAP2K1, in which the genetically induced gradual deterioration of bone microarchitecture triggers a periosteal reaction, similar to the process found to occur after bone infection or local trauma, and leads to an overall cortical outgrowth. The micromechanical properties of the lesions reflect their structural heterogeneity and correlate with local variations in mineral content, tissue age, and remodeling rates, in the same way as normal bone. © 2018 American Society for Bone and Mineral Research.
Asunto(s)
Densidad Ósea , MAP Quinasa Quinasa 1 , Melorreostosis , Modelos Biológicos , Mutación , Periostio , Microtomografía por Rayos X , Adulto , Femenino , Humanos , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Masculino , Melorreostosis/diagnóstico por imagen , Melorreostosis/genética , Melorreostosis/metabolismo , Melorreostosis/fisiopatología , Persona de Mediana Edad , Periostio/diagnóstico por imagen , Periostio/metabolismo , Periostio/fisiopatologíaRESUMEN
Melorheostosis is a sporadic disease of uncertain etiology characterized by asymmetric bone overgrowth and functional impairment. Using whole exome sequencing, we identify somatic mosaic MAP2K1 mutations in affected, but not unaffected, bone of eight unrelated patients with melorheostosis. The activating mutations (Q56P, K57E and K57N) cluster tightly in the MEK1 negative regulatory domain. Affected bone displays a mosaic pattern of increased p-ERK1/2 in osteoblast immunohistochemistry. Osteoblasts cultured from affected bone comprise two populations with distinct p-ERK1/2 levels by flow cytometry, enhanced ERK1/2 activation, and increased cell proliferation. However, these MAP2K1 mutations inhibit BMP2-mediated osteoblast mineralization and differentiation in vitro, underlying the markedly increased osteoid detected in affected bone histology. Mosaicism is also detected in the skin overlying bone lesions in four of five patients tested. Our data show that the MAP2K1 oncogene is important in human bone formation and implicate MEK1 inhibition as a potential treatment avenue for melorheostosis.
Asunto(s)
Huesos/metabolismo , MAP Quinasa Quinasa 1/genética , Melorreostosis/genética , Mutación , Osteoblastos/metabolismo , Osteogénesis/genética , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Huesos/patología , Calcificación Fisiológica , Diferenciación Celular , Proliferación Celular , Regulación de la Expresión Génica , Humanos , MAP Quinasa Quinasa 1/metabolismo , Melorreostosis/metabolismo , Melorreostosis/patología , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mosaicismo , Osteoblastos/patología , Cultivo Primario de Células , Transducción de Señal , Piel/metabolismo , Piel/patología , Secuenciación del ExomaRESUMEN
Cyclophilin B (CypB) is an endoplasmic reticulum-resident protein that regulates collagen folding, and also contributes to prolyl 3-hydroxylation (P3H) and lysine (Lys) hydroxylation of collagen. In this study, we characterized dentin type I collagen in CypB null (KO) mice, a model of recessive osteogenesis imperfecta type IX, and compared to those of wild-type (WT) and heterozygous (Het) mice. Mass spectrometric analysis demonstrated that the extent of P3H in KO collagen was significantly diminished compared to WT/Het. Lys hydroxylation in KO was significantly diminished at the helical cross-linking sites, α1/α2(I) Lys-87 and α1(I) Lys-930, leading to a significant increase in the under-hydroxylated cross-links and a decrease in fully hydroxylated cross-links. The extent of glycosylation of hydroxylysine residues was, except α1(I) Lys-87, generally higher in KO than WT/Het. Some of these molecular phenotypes were distinct from other KO tissues reported previously, indicating the dentin-specific control mechanism through CypB. Histological analysis revealed that the width of predentin was greater and irregular, and collagen fibrils were sparse and significantly smaller in KO than WT/Het. These results indicate a critical role of CypB in dentin matrix formation, suggesting a possible association between recessive osteogenesis imperfecta and dentin defects that have not been clinically detected.
Asunto(s)
Colágeno Tipo I , Ciclofilinas/deficiencia , Dentina/ultraestructura , Animales , Colágeno Tipo I/ultraestructura , Ciclofilinas/fisiología , Dentina/patología , Matriz Extracelular/patología , Matriz Extracelular/ultraestructura , Glicosilación , Hidroxilación , Lisina/metabolismo , Espectrometría de Masas , Ratones , Ratones Noqueados , Osteogénesis Imperfecta , Procolágeno-Prolina Dioxigenasa/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Context: Recessive mutations in TMEM38B cause type XIV osteogenesis imperfecta (OI) by dysregulating intracellular calcium flux. Objectives: Clinical and bone material phenotype description and osteoblast differentiation studies. Design and Setting: Natural history study in pediatric research centers. Patients: Eight patients with type XIV OI. Main Outcome Measures: Clinical examinations included bone mineral density, radiographs, echocardiography, and muscle biopsy. Bone biopsy samples (n = 3) were analyzed using histomorphometry, quantitative backscattered electron microscopy, and Raman microspectroscopy. Cellular differentiation studies were performed on proband and control osteoblasts and normal murine osteoclasts. Results: Type XIV OI clinical phenotype ranges from asymptomatic to severe. Previously unreported features include vertebral fractures, periosteal cloaking, coxa vara, and extraskeletal features (muscular hypotonia, cardiac abnormalities). Proband lumbar spine bone density z score was reduced [median -3.3 (range -4.77 to +0.1; n = 7)] and increased by +1.7 (1.17 to 3.0; n = 3) following bisphosphonate therapy. TMEM38B mutant bone has reduced trabecular bone volume, osteoblast, and particularly osteoclast numbers, with >80% reduction in bone resorption. Bone matrix mineralization is normal and nanoporosity low. We demonstrate a complex osteoblast differentiation defect with decreased expression of early markers and increased expression of late and mineralization-related markers. Predominance of trimeric intracellular cation channel type B over type A expression in murine osteoclasts supports an intrinsic osteoclast defect underlying low bone turnover. Conclusions: OI type XIV has a bone histology, matrix mineralization, and osteoblast differentiation pattern that is distinct from OI with collagen defects. Probands are responsive to bisphosphonates and some show muscular and cardiovascular features possibly related to intracellular calcium flux abnormalities.
Asunto(s)
Coxa Vara/fisiopatología , Canales Iónicos/genética , Osteoblastos/fisiología , Osteoclastos/fisiología , Osteogénesis Imperfecta/fisiopatología , Fracturas de la Columna Vertebral/fisiopatología , Adolescente , Adulto , Animales , Densidad Ósea , Calcio/metabolismo , Hueso Esponjoso/diagnóstico por imagen , Hueso Esponjoso/patología , Estudios de Casos y Controles , Recuento de Células , Diferenciación Celular , Niño , Preescolar , Coxa Vara/etiología , Ecocardiografía , Femenino , Perfilación de la Expresión Génica , Genotipo , Cardiopatías/diagnóstico por imagen , Cardiopatías/etiología , Cardiopatías/fisiopatología , Heterocigoto , Humanos , Lactante , Recién Nacido , Canales Iónicos/metabolismo , Vértebras Lumbares/diagnóstico por imagen , Masculino , Ratones , Microscopía Electrónica , Hipotonía Muscular/etiología , Hipotonía Muscular/fisiopatología , Mutación , Tamaño de los Órganos , Osteoblastos/citología , Osteoclastos/citología , Osteogénesis Imperfecta/complicaciones , Osteogénesis Imperfecta/diagnóstico por imagen , Osteogénesis Imperfecta/genética , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la Enfermedad , Espectrometría Raman , Fracturas de la Columna Vertebral/etiología , Adulto JovenRESUMEN
Limited detoxification capacity often directs aggregation-prone, potentially hazardous, misfolded proteins to be deposited in designated cytosolic compartments known as 'aggresomes'. The roles of aggresomes as cellular quality control centers, and the cellular origin of the deposits contained within these structures, remain to be characterized. Here, we utilized the observation that the prion protein (PrP, also known as PRNP) accumulates in aggresomes following the inhibition of folding chaperones, members of the cyclophilin family, to address these questions. We found that misfolded PrP molecules must pass through the endoplasmic reticulum (ER) in order to be deposited in aggresomes, that the Golgi plays no role in this process and that cytosolic PrP species are not deposited in pre-existing aggresomes. Prior to their deposition in the aggresome, PrP molecules lose the ER localization signal and have to acquire a GPI anchor. Our discoveries indicate that PrP aggresomes are cytosolic overflow deposition centers for the ER quality control mechanisms and highlight the importance of these structures for the maintenance of protein homeostasis within the ER.
Asunto(s)
Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Priónicas/metabolismo , Agregado de Proteínas , Animales , Células CHO , Cricetinae , Cricetulus , Ciclosporina/farmacología , Citosol/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Glicosilación , Glicosilfosfatidilinositoles/metabolismo , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Agregado de Proteínas/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacosRESUMEN
Osteogenesis imperfecta (OI) is a collagen-related bone dysplasia. We identified an X-linked recessive form of OI caused by defects in MBTPS2, which encodes site-2 metalloprotease (S2P). MBTPS2 missense mutations in two independent kindreds with moderate/severe OI cause substitutions at highly conserved S2P residues. Mutant S2P has normal stability, but impaired functioning in regulated intramembrane proteolysis (RIP) of OASIS, ATF6 and SREBP transcription factors, consistent with decreased proband secretion of type I collagen. Further, hydroxylation of the collagen lysine residue (K87) critical for crosslinking is reduced in proband bone tissue, consistent with decreased lysyl hydroxylase 1 in proband osteoblasts. Reduced collagen crosslinks presumptively undermine bone strength. Also, proband osteoblasts have broadly defective differentiation. These mutations provide evidence that RIP plays a fundamental role in normal bone development.
Asunto(s)
Membrana Celular/patología , Colágeno Tipo I/genética , Metaloendopeptidasas/genética , Mutación Missense , Osteoblastos/metabolismo , Osteogénesis Imperfecta/genética , Factor de Transcripción Activador 6/genética , Factor de Transcripción Activador 6/metabolismo , Adulto , Anciano , Diferenciación Celular , Membrana Celular/metabolismo , Colágeno Tipo I/deficiencia , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Genes Recesivos , Humanos , Hidroxilación , Masculino , Metaloendopeptidasas/metabolismo , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Osteoblastos/patología , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/patología , Linaje , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Proteolisis , Índice de Severidad de la Enfermedad , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50-70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes.
Asunto(s)
Calcio/metabolismo , Colágeno Tipo I/biosíntesis , Canales Iónicos/genética , Osteogénesis Imperfecta/genética , Adulto , Señalización del Calcio , Colágeno Tipo I/metabolismo , Consanguinidad , Análisis Mutacional de ADN , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Femenino , Genes Recesivos , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Homeostasis , Humanos , Lactante , Masculino , Linaje , Procesamiento Proteico-PostraduccionalRESUMEN
Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1-3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation.
Asunto(s)
Colágeno Tipo I/química , Lisina/química , Animales , Colágeno/química , Ciclofilinas/metabolismo , Hidroxilación , Tendones/metabolismoRESUMEN
Do different neurodegenerative maladies emanate from the failure of a mutual protein folding mechanism? We have addressed this question by comparing mutational patterns that are linked to the manifestation of distinct neurodegenerative disorders and identified similar neurodegeneration-linked proline substitutions in the prion protein and in presenilin 1 that underlie the development of a prion disorder and of familial Alzheimer's disease (fAD), respectively. These substitutions were found to prevent the endoplasmic reticulum (ER)-resident chaperone, cyclophilin B, from assisting presenilin 1 to fold properly, leading to its aggregation, deposition in the ER, reduction of γ-secretase activity, and impaired mitochondrial distribution and function. Similarly, reduced quantities of the processed, active presenilin 1 were observed in brains of cyclophilin B knockout mice. These discoveries imply that reduced cyclophilin activity contributes to the development of distinct neurodegenerative disorders, propose a novel mechanism for the development of certain fAD cases, and support the emerging theme that this disorder can stem from aberrant presenilin 1 function. This study also points at ER chaperones as targets for the development of counter-neurodegeneration therapies.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Sustitución de Aminoácidos , Encéfalo/metabolismo , Presenilina-1/metabolismo , Agregación Patológica de Proteínas/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Encéfalo/patología , Línea Celular , Ratones , Ratones Noqueados , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Presenilina-1/genética , Prolina/genética , Prolina/metabolismo , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , Pliegue de ProteínaRESUMEN
CONTEXT: Patients with type V osteogenesis imperfecta (OI) are heterozygous for a dominant IFITM5 c.-14C>T mutation, which adds five residues to the N terminus of bone-restricted interferon-induced transmembrane-like protein (BRIL), a transmembrane protein expressed in osteoblasts. Type V OI skeletal findings include hyperplastic callus formation, ossification of the forearm interosseous membrane, and dense metaphyseal bands. OBJECTIVE: The objective of this study was to examine the role of osteoblasts in the active mineralization traits of type V OI and the effect of the IFITM5 mutation on type I collagen. METHODS: We identified eight patients with the IFITM5 c.-14C>T mutation. Cultured osteoblasts from type V OI patients were used to study osteoblast differentiation and mineralization. RESULTS: We verified the expression and stability of mutant IFITM5 transcripts. In differentiated type V OI primary osteoblasts in culture, the IFITM5 expression and BRIL level is comparable with control. Both early and late markers of osteoblast differentiation are increased in type V OI osteoblasts. Mineralization, assayed by alizarin red staining, was increased in type V OI osteoblasts compared with control. However, type V OI osteoblasts have significantly decreased COL1A1 transcripts in mid- to late differentiation. Type I collagen protein is concomitantly decreased, with decreased cross-linked collagen in matrix and altered appearance of fibrils deposited in culture. CONCLUSIONS: This study demonstrates that type V OI mineralization has a gain-of-function mechanism at the osteoblast level, which likely underlies the overactive tissue mineralization seen in patients. Decreased type I collagen expression, secretion, and matrix incorporation establish type V OI as a collagen-related defect.
Asunto(s)
Calcinosis/patología , Colágeno Tipo I/genética , Proteínas de la Membrana/genética , Osteoblastos/patología , Osteogénesis Imperfecta/patología , Adulto , Anciano , Calcinosis/genética , Calcinosis/metabolismo , Preescolar , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Femenino , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Mutación , Osteoblastos/metabolismo , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/metabolismo , Adulto JovenRESUMEN
Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink pattern was associated with decreased collagen deposition into matrix in culture, altered fibril structure in tissue, and reduced bone strength. These studies demonstrate novel consequences of the indirect regulatory effect of CyPB on collagen hydroxylation, impacting collagen glycosylation, crosslinking and fibrillogenesis, which contribute to maintaining bone mechanical properties.