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1.
J Cell Biochem ; 118(10): 3328-3340, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28295540

RESUMEN

Vacuolar H+ -ATPases (V-ATPases) are ubiquitous multisubunit proton pumps responsible for organellar pH maintenance. Mutations in the a3 subunit of V-ATPases cause autosomal recessive osteopetrosis, a rare disease due to impaired bone resorption. Patients with osteopetrosis also display dental anomalies, such as enamel defects; however, it is not clear whether these enamel abnormalities are a direct consequence of the a3 mutations. We investigated enamel mineralization, spatiotemporal expression of enamel matrix proteins and the a3 protein during tooth development using an osteopetrotic mouse model with a R740S point mutation in the V-ATPase a3 subunit. Histology revealed aberrations in both crown and root development, whereas SEM analysis demonstrated delayed enamel mineralization in homozygous animals. Enamel thickness and mineralization were significantly decreased in homozygous mice as determined by µCT analysis. The expression patterns of the enamel matrix proteins amelogenin, amelotin, and odontogenic ameloblast-associated protein (ODAM) suggested a delay in transition to the maturation stage in homozygous animals. Protein expression of the a3 subunit was detected in ameloblasts in all three genotypes, suggesting that a3-containing V-ATPases play a direct role in amelogenesis, and mutations in a3 delay transition from the secretory to the maturation stage, resulting in hypomineralized and hypoplastic enamel. J. Cell. Biochem. 118: 3328-3340, 2017. © 2017 Wiley Periodicals, Inc.


Asunto(s)
Calcificación Fisiológica/fisiología , Esmalte Dental/enzimología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Esmalte Dental/crecimiento & desarrollo , Ratones , Ratones Mutantes , Osteopetrosis/enzimología , Osteopetrosis/genética , Mutación Puntual , ATPasas de Translocación de Protón Vacuolares/genética
2.
J Cell Biochem ; 117(2): 413-25, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26212375

RESUMEN

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in the regulation of cell growth. It has been shown to play an important role in osteoclast differentiation, particularly at the earlier stages of osteoclastogenesis. mTOR activation and function, as part of mTORC1 complex, is dependent on lysosomal localization and the vacuolar H(+) -ATPase (V-ATPase) activity; however, the precise mechanism is still not well understood. Using primary mouse osteoclasts that are known to have higher lysosomal pH due to R740S mutation in the V-ATPase a3 subunit, we investigated the role of lysosomal pH in mTORC1 signaling. Our results demonstrated that +/R740S cells had increased basal mTOR protein levels and mTORC1 activity compared to +/+ osteoclasts, while mTOR gene expression was decreased. Treatment with lysosomal inhibitors chloroquine and ammonium chloride, compounds known to raise lysosomal pH, significantly increased mTOR protein levels in +/+ cells, confirming the importance of lysosomal pH in mTOR signaling. These results also suggested that mTOR could be degraded in the lysosome. To test this hypothesis, we cultured osteoclasts with chloroquine or proteasomal inhibitor MG132. Both chloroquine and MG132 increased mTOR and p-mTOR protein levels in +/+ osteoclasts, suggesting that mTOR undergoes both lysosomal and proteasomal degradation. Treatment with cycloheximide, an inhibitor of new protein synthesis, confirmed that mTOR is constitutively expressed and degraded. These results show that, in osteoclasts, the lysosome plays a key role not only in mTOR activation but also in its deactivation through protein degradation, representing a novel molecular mechanism of mTOR regulation.


Asunto(s)
Lisosomas/metabolismo , Osteoclastos/enzimología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Autofagia , Células Cultivadas , Activación Enzimática , Expresión Génica , Concentración de Iones de Hidrógeno , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones Endogámicos C3H , Ratones Transgénicos , Complejos Multiproteicos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transporte de Proteínas , Proteolisis , Serina-Treonina Quinasas TOR/genética
3.
J Cell Biochem ; 114(12): 2823-33, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23908015

RESUMEN

Vacuolar-type H(+)-ATPases (V-ATPases) are located in lysosomes and at the ruffled border in osteoclasts. We showed previously that the R740S mutation is dominant negative for V-ATPase activity, uncouples proton transport from ATP hydrolysis and causes osteopetrosis in heterozygous mice (+/R740S). Here we show mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis and die by postnatal day 14. Although R740S/R740S osteoclasts express wild-type levels of a3, it is mislocalized. Acridine orange staining of R740S/R740S osteoclasts grown on a Corning resorptive surface reveals no resorption and no acidification of intracellular compartments. Whereas osteoblast and osteocyte apoptosis is normal, R740S/R740S osteoclasts exhibit increased apoptosis compared with wild-type osteoclasts. Localization of the enzyme tartrate-resistant acid phosphatase (TRAP) is also aberrant. Transmission electron microscopy reveals that R740S/R740S osteoclasts do not polarize, lack ruffled borders, and contain fewer autophagosomes. Consistent with an early stage defect in autophagy, expression of LC3II is reduced and expression of p62 is increased in R740S/R740S compared to wild-type osteoclasts. These results indicate the importance of intracellular acidification for the early stages of autophagy as well as for osteoclast survival, maturation, and polarization with appropriate cytoplasmic distribution of key osteoclast enzymes such as TRAP.


Asunto(s)
Osteoclastos/citología , Osteopetrosis/genética , Subunidades de Proteína/genética , ATPasas de Translocación de Protón Vacuolares/genética , Animales , Apoptosis/genética , Autofagia/genética , Diferenciación Celular/genética , Citoplasma/genética , Lisosomas/genética , Lisosomas/metabolismo , Ratones , Mutación , Osteoclastos/metabolismo , Osteopetrosis/enzimología , Osteopetrosis/patología , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo
4.
J Cell Biochem ; 113(7): 2432-41, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22573557

RESUMEN

X-linked hypophosphatemic rickets (XLH) is a dominantly inherited disease characterized by renal phosphate wasting, aberrant vitamin D metabolism, and defective bone mineralization. It is known that XLH in humans and in certain mouse models is caused by inactivating mutations in PHEX/Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). By a genome-wide N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a dominant mouse mutation that exhibits the classic clinical manifestations of XLH, including growth retardation, skeletal abnormalities (rickets/osteomalacia), hypophosphatemia, and increased serum alkaline phosphatase (ALP) levels. Mapping and sequencing revealed that these mice carry a point mutation in exon 14 of the Phex gene that introduces a stop codon at amino acid 496 of the coding sequence (Phex(Jrt) also published as Phex(K496X) [Ichikawa et al., 2012]). Fgf23 mRNA expression as well as that of osteocalcin, bone sialoprotein, and matrix extracellular phosphoglycoprotein was upregulated in male mutant long bone, but that of sclerostin was unaffected. Although Phex mRNA is expressed in bone from mutant hemizygous male mice (Phex(Jrt)/Y mice), no Phex protein was detected in immunoblots of femoral bone protein. Stromal cultures from mutant bone marrow were indistinguishable from those of wild-type mice with respect to differentiation and mineralization. The ability of Phex(Jrt)/Y osteoblasts to mineralize and the altered expression levels of matrix proteins compared with the well-studied Hyp mice makes it a unique model with which to further explore the clinical manifestations of XLH and its link to FGF23 as well as to evaluate potential new therapeutic strategies.


Asunto(s)
Huesos/patología , Modelos Animales de Enfermedad , Raquitismo Hipofosfatémico Familiar , Enfermedades Genéticas Ligadas al Cromosoma X , Endopeptidasa Neutra Reguladora de Fosfato PHEX/genética , Mutación Puntual , Proteínas Adaptadoras Transductoras de Señales , Animales , Secuencia de Bases , Células de la Médula Ósea , Huesos/metabolismo , Calcificación Fisiológica/genética , Calcificación Fisiológica/fisiología , Células Cultivadas , Mapeo Cromosómico , Etilnitrosourea , Proteínas de la Matriz Extracelular/biosíntesis , Raquitismo Hipofosfatémico Familiar/genética , Raquitismo Hipofosfatémico Familiar/metabolismo , Raquitismo Hipofosfatémico Familiar/patología , Femenino , Factor-23 de Crecimiento de Fibroblastos , Glicoproteínas/biosíntesis , Sialoproteína de Unión a Integrina/biosíntesis , Péptidos y Proteínas de Señalización Intercelular , Masculino , Ratones , Ratones Endogámicos C57BL , Mutágenos/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/biosíntesis , Fosfoproteínas/biosíntesis , ARN Mensajero/biosíntesis , Análisis de Secuencia de ADN , Células del Estroma
5.
Sci Rep ; 9(1): 11211, 2019 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-31371754

RESUMEN

Oculocutaneous syndromes are often due to mutations in single genes. In some cases, mouse models for these diseases exist in spontaneously occurring mutations, or in mice resulting from forward mutatagenesis screens. Here we present novel genes that may be causative for oculocutaneous disease in humans, discovered as part of a genome-wide screen of knockout-mice in a targeted single-gene deletion project. The International Mouse Phenotyping Consortium (IMPC) database (data release 10.0) was interrogated for all mouse strains with integument abnormalities, which were then cross-referenced individually to identify knockouts with concomitant ocular abnormalities attributed to the same targeted gene deletion. The search yielded 307 knockout strains from unique genes with integument abnormalities, 226 of which have not been previously associated with oculocutaneous conditions. Of the 307 knockout strains with integument abnormalities, 52 were determined to have ocular changes attributed to the targeted deletion, 35 of which represent novel oculocutaneous genes. Some examples of various integument abnormalities are shown, as well as two examples of knockout strains with oculocutaneous phenotypes. Each of the novel genes provided here are potentially relevant to the pathophysiology of human integumentary, or oculocutaneous conditions, such as albinism, phakomatoses, or other multi-system syndromes. The novel genes reported here may implicate molecular pathways relevant to these human diseases and may contribute to the discovery of novel therapeutic targets.


Asunto(s)
Albinismo Oculocutáneo/genética , Integumento Común/anomalías , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Pigmentación/genética
7.
Commun Biol ; 1: 236, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30588515

RESUMEN

Despite advances in next generation sequencing technologies, determining the genetic basis of ocular disease remains a major challenge due to the limited access and prohibitive cost of human forward genetics. Thus, less than 4,000 genes currently have available phenotype information for any organ system. Here we report the ophthalmic findings from the International Mouse Phenotyping Consortium, a large-scale functional genetic screen with the goal of generating and phenotyping a null mutant for every mouse gene. Of 4364 genes evaluated, 347 were identified to influence ocular phenotypes, 75% of which are entirely novel in ocular pathology. This discovery greatly increases the current number of genes known to contribute to ophthalmic disease, and it is likely that many of the genes will subsequently prove to be important in human ocular development and disease.

8.
J Bone Miner Res ; 22(9): 1373-86, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17547535

RESUMEN

UNLABELLED: We performed a longitudinal analysis of bone quality in Sca-1-null mice. A tight temporal, site-specific association between Sca-1-deficient BMD deficiency and reduced mesenchymal progenitor frequency was observed. Defects in trabecular microarchitecture and mineralization were, at least partially, responsible for the age-related reduction in toughness of Sca-1(-/-) bones. INTRODUCTION: We previously showed that stem cell antigen 1 (Sca-1)-null mice undergo normal bone development but exhibit significantly decreased bone mass characteristic of age-dependent osteoporosis. The objective of this study was to characterize the initiation and progression of the Sca-1 mutant skeletal phenotype at the cellular, structural, material, and mechanical levels. MATERIALS AND METHODS: Sca-1-null and control mice were analyzed at 3, 5, 7, and 9 mo of age. In vitro osteoclastogenesis of bone marrow and spleen-derived progenitor populations was assessed. Bone marrow-derived mesenchymal progenitor frequency, along with osteogenic and adipogenic differentiation potential, was analyzed in vitro. Static histomorphometry of the sixth lumbar vertebrae was performed. Whole body, femoral, and vertebral BMD were assessed using DXA. Lumbar vertebrae were analyzed using microCT, back-scattered electron imaging, and compression tests. Three-point bending and femoral neck fracture tests were performed on excised femurs. RESULTS: Sca-1-null mice displayed an age-dependent, cell-autonomous osteoclast deficiency in vitro. From 7 mo of age onward, reduced Sca-1-null femoral BMD was observed alongside reduced mesenchymal progenitor frequency, and decreased in vitro osteogenic and adipogenic differentiation potential. Sca-1-deficient mice exhibited reduced whole body BMD compared with controls at all time-points analyzed. Although no differences in spinal BMD were observed, Sca-1(-/-) vertebrae exhibited decreased bone formation, with a maximal difference at 7 mo of age, inferior trabecular microarchitecture, and a greater degree of mineralization. At all sites tested, Sca-1-null bones exhibited reduced energy to failure from 5 mo onward. CONCLUSIONS: We showed a tight association within Sca-1-null mice between the initiation of stem cell defects and the exacerbation of deficiencies in bone quality at two sites clinically relevant to developing osteoporotic fractures. Sca-1-deficient mice, therefore, provide a novel and useful murine model of age-related osteoporosis, which with additional study, should further our understanding of the mechanisms underlying this increasingly prevalent disease.


Asunto(s)
Antígenos Ly/fisiología , Huesos/patología , Proteínas de la Membrana/fisiología , Células Madre Mesenquimatosas/citología , Osteoporosis/patología , Tejido Adiposo/patología , Animales , Antígenos Ly/genética , Calcificación Fisiológica , Células Cultivadas , Femenino , Estudios Longitudinales , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Osteoclastos/citología
9.
Sci Rep ; 7(1): 3005, 2017 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-28592812

RESUMEN

Mammalian target of rapamycin (mTOR) is activated by numerous stimuli, including amino acids and growth factors. This kinase is part of the mTOR complex 1 (mTORC1) which regulates cell proliferation, differentiation, and autophagy. Active mTORC1 is located on lysosomes and has been reported to disassociate from the lysosomal surface in the absence of amino acids. Furthermore, mTORC1 activity has been linked to the vacuolar H+-ATPases (V-ATPases), the proton pumps responsible for lysosomal acidification; however, the exact role of the V-ATPases in mTORC1 signaling is not known. To elucidate the mechanisms involved in mTORC1 regulation by the V-ATPases, we used primary osteoclasts derived from mice carrying a point (R740S) mutation in the a3 subunit of the V-ATPase. In these cells, the mutant protein is expressed but the pump is not functional, resulting in higher lysosomal pH. By analyzing mTOR activation, mTOR/lysosome co-localization, and lysosomal positioning using confocal microscopy, fractionation, and ultrapure lysosomal purification methods, we demonstrate that in primary osteoclasts, mTOR is localized on the lysosomal surface even when mTOR activity is inhibited. Our findings reveal that mTOR targeting to the lysosome in osteoclasts is activity-independent, and that its disassociation from the lysosome during starvation is not universal.


Asunto(s)
Lisosomas/metabolismo , Osteoclastos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Fraccionamiento Celular , Células Cultivadas , Ratones , Microscopía Confocal , Proteínas Mutantes/metabolismo , Transporte de Proteínas , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo
10.
J Bone Miner Res ; 28(1): 108-18, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22865292

RESUMEN

Vacuolar H(+) -ATPase (V-ATPase), a multisubunit enzyme located at the ruffled border and in lysosomes of osteoclasts, is necessary for bone resorption. We previously showed that heterozygous mice with an R740S mutation in the a3 subunit of V-ATPase (+/R740S) have mild osteopetrosis resulting from an ∼90% reduction in proton translocation across osteoclast membranes. Here we show that lysosomal pH is also higher in +/R740S compared with wild-type (+/+) osteoclasts. Both osteoclast number and size were decreased in cultures of +/R740S compared with +/+ bone marrow cells, with concomitant decreased expression of key osteoclast markers (TRAP, cathepsin K, OSCAR, DC-STAMP, and NFATc1), suggesting that low lysosomal pH plays an important role in osteoclastogenesis. To elucidate the molecular mechanism of this inhibition, NFATc1 activation was assessed. NFATc1 nuclear translocation was significantly reduced in +/R740S compared with +/+ cells; however, this was not because of impaired enzymatic activity of calcineurin, the phosphatase responsible for NFATc1 dephosphorylation. Protein and RNA expression levels of regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of NFATc1 activation and a protein degraded in lysosomes, were not significantly different between +/R740S and +/+ osteoclasts, but the RCAN1/NFATc1 ratio was significantly higher in +/R740S versus +/+ cells. The lysosomal inhibitor chloroquine significantly increased RCAN1 accumulation in +/+ cells, consistent with the hypothesis that higher lysosomal pH impairs RCAN1 degradation, leading to a higher RCAN1/NFATc1 ratio and consequently NFATc1 inhibition. Our data indicate that increased lysosomal pH in osteoclasts leads to decreased NFATc1 signaling and nuclear translocation, resulting in a cell autonomous impairment of osteoclastogenesis in vitro.


Asunto(s)
Sustitución de Aminoácidos/genética , Lisosomas/metabolismo , Mutación/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Osteogénesis , ATPasas de Translocación de Protón Vacuolares/genética , Animales , Biomarcadores/metabolismo , Calcineurina/metabolismo , Proteínas de Unión al Calcio , Regulación de la Expresión Génica , Concentración de Iones de Hidrógeno , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Proteínas Musculares/metabolismo , Factores de Transcripción NFATC/antagonistas & inhibidores , Osteoclastos/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Proteínas Tirosina Quinasas/metabolismo , Quinasas DyrK
11.
J Bone Miner Res ; 26(7): 1484-93, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21305608

RESUMEN

A mouse founder with high bone mineral density and an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) screen. It was found to carry a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit of the vacuolar type H(+)-ATPase (V-ATPase), resulting in replacement of a highly conserved amino acid (R740S). The +/R740S mice have normal appearance, size, and weight but exhibit high bone density. Osteoblast parameters are unaffected in bones of +/R740S mice, whereas osteoclast number and marker expression are increased, concomitant with a decrease in the number of apoptotic osteoclasts. Consistent with reduced osteoclast apoptosis, expression of Rankl and Bcl2 is elevated, whereas Casp3 is reduced. Transmission electron microscopy revealed that unlike other known mutations in the a3 subunit of V-ATPase, polarization and ruffled border formation appear normal in +/R740S osteoclasts. However, V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport, whereas ATP hydrolysis is not significantly affected. We show for the first time that a point mutation within the a3 subunit, R740S, which is dominant negative for proton pumping and bone resorption, also uncouples proton pumping from ATP hydrolysis but has no effect on ruffled border formation or polarization of osteoclasts. These results suggest that the V(0) complex has proton-pumping-independent functions in mammalian cells.


Asunto(s)
Sustitución de Aminoácidos/genética , Genes Dominantes/genética , Mutación Missense/genética , Osteopetrosis/genética , Subunidades de Proteína/genética , ATPasas de Translocación de Protón Vacuolares/genética , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Secuencia de Bases , Transporte Biológico , Recuento de Células , Forma de la Célula , Análisis Mutacional de ADN , Femenino , Fémur/diagnóstico por imagen , Fémur/patología , Heterocigoto , Hidrólisis , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/metabolismo , Osteoclastos/patología , Osteopetrosis/patología , Protones , Microtomografía por Rayos X
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